University Department of Obstetrics & Gynaecology, Royal Infirmary, Glasgow
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Abstract |
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Key words: follicle stimulating hormone/follicular growth/gonadotrophins/luteinising hormone/ovarian stimulation
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Introduction |
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In the maturing Graafian follicle, the role of LH is governed by the two-cell, two-gonadotrophin hypothesis, whereby LH induces androgen biosynthesis in the theca cells, which the FSH-stimulated granulosa cells convert to oestrogen. This accounts for the appearance of oestradiol in the follicular fluid and the peripheral circulation, and an absence of measurable LH leads to negligible oestradiol production, even when FSH can stimulate follicular growth (Couzinet et al., 1988). In fact, it has been proposed that the latter stages of follicular growth in women, may be induced by FSH alone (Loumaye et al., 1997
). However, the results of a recent multicentre study with recombinant FSH (rFSH) and recombinant LH (rLH) treatment of women with hypogonadotrophic hypogonadism, suggested that LH actually sensitizes follicles to FSH, and thereby encourages follicular development as well as hormone secretion (ERHLHSG, 1998
). The number of follicles developing after a fixed dose of rFSH was much reduced or delayed in the group treated without rLH. The dose of 75 IU rLH per day appeared to be sufficient to facilitate most functions of follicular development.
A role of LH at other levels of ovarian function may be implied by two other lines of evidence. In an in-vitro small mammal model, the importance of the interaction between LH and the theca cell mass in physiological events as well as follicular endocrinology has been highlighted (Cortvrindt et al., 1998). Mouse follicles cultured in the presence of rFSH without rLH clearly demonstrated a reduced rate of antrum formation, and oocyte nuclear maturation. In sheep follicular cells and intact follicles cultured in vitro, LH was seen to be obligatory for the production of specific polypeptides seen in vivo (Moor and Crosby, 1987
).
Further evidence derives from a woman with hypogonadotrophic hypogonadism undergoing IVF after treatment with purified FSH or FSH combined with additional oestradiol, in which oocytes were obtained which showed poor fertilization rates. The same individual was subsequently treated with combined FSH and LH (human menopausal gonadotrophin; HMG), and the oocytes showed normal fertilization rates. These data suggest that the LH-depleted environment may yield oocytes with a reduced potential for further development (Balasch et al., 1995).
Further evidence for LH activity during the follicular phase having an impact upon oocyte/embryo competence was provided by studies in macaque monkeys in which LH was suppressed using a gonadotrophin-releasing hormone antagonist, and follicular growth stimulated with rFSH or rFSH with rLH. Reasonable follicular growth and oocyte yields were obtained after stimulation with rFSH alone, and higher fertilization rates were obtained with rFSH compared with the combination of rFSH and rLH (Zelinski-Wooten et al., 1996). However, the absence of rLH had a detrimental effect upon embryo cryosurvival, development and implantation after cryopreservation and thawing (Weston et al., 1996
). These data imply that there may be an undefined physiological role of LH with implications for oocyte maturation and embryo developmental potential.
In the clinical situation of assisted reproduction, the three most important criteria are numbers of oocytes obtained after a course of ovarian stimulation, their developmental competence, and the ability of implanted embryos to sustain viable fetal development.
It is clear that the widespread use of urinary FSH or purified urinary FSH, without LH supplementation in ovarian stimulation, has not led to a drop in overall programme success rates, and it has been suggested that there is no need to explore the requirement for LH supplementation further (Hull et al., 1994). However, different hormone profiles have been recorded according to LH activity in the stimulant and/or in the circulation (Fleming et al., 1996
; Fried et al., 1996
), and the exploration of other possible effects of LH depletion after gonadotrophin-releasing hormone analogue (GnRHa) co-treatment have been rare. Two prospective studies were performed to examine the effects of profoundly suppressed LH during the follicular phase of COS cycles. The results indicated that when purified, urinary FSH was used, profound suppression of LH had no effect upon oocyte or embryo developmental potential, but there were implications with respect to ovarian steroid and inhibin biosynthesis, and also a reduced oocyte and embryo yield (Fleming et al., 1998
). One of the aims of this study was to determine if effects seen in a relatively small and controlled prospective study could be detected in an unselected evaluation within a treatment programme, where the population shows much greater diversity.
Recently, rFSH has also enjoyed widespread use in ovarian stimulation programmes with considerable success. It has different isoform profiles from urinary-derived FSH and is probably, thereby, more potent (De Leeuw et al., 1996) and appears to show increased efficacy and improved pregnancy rates by comparison with purified FSH treatment (Out et al., 1995
). However, the role of profoundly suppressed follicular phase LH during COS has not been determined with rFSH as the ovarian stimulant. Differences in responses with respect to LH in the circulation which are influenced by FSH source would indicate that in this clinical situation, there is a balance of importance between FSH potency and the biochemical roles of LH.
In response to these observations an investigation was undertaken exploring the effects of profound LH suppression in two complete databases of patients in the assisted conception programme treated with purified, urinary FSH or with rFSH. The two databases represented separate cohorts of patients with no selectivity bias. Chronological differences dictated that data could only be compared within databases, not between them.
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Materials and methods |
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All were treated with the standard long course GnRHa treatment starting on cycle day 21, or, in women with oligomenorrhoea, at least 14 days prior to initiation of gonadotrophin stimulation. The normal starting dose of FSH for ovarian stimulation was 225 IU daily, unless otherwise indicated by previous experience, and response assessment effected on stimulation day 7 (S7), when dose titration was effected. If the oestradiol concentration was <200 pg/ml the dose was increased, whereas if the oestradiol value was >1000 pg/ml the dose was reduced to 150 IU. The luteinizing signal of human chorionic gonadotrophin (HCG, Profasi; Serono UK Ltd, Welwyn Garden City, UK: 10 000 IU) was administered only when there were more than two mature sized follicles (diameter >16 mm) observed by ultrasonography. Oocyte retrieval was effected 3740 h after HCG administration. Cycles were cancelled for `poor response' after 5 days continued treatment with the increased dose without attaining, or the imminent prospect of attaining, the criteria for HCG administration.
Embryo cryopreservation was effected when there were more than one `good quality' embryos available after the fresh embryo transfer. In order to reduce the risks and consequences of ovarian hyperstimulation syndrome, cases with >16 oocytes recovered did not receive a fresh embryo transfer, as all embryos were cryopreserved for transfer in a subsequent cycle.
Treatment and hormone assays
All cycles of ovarian stimulation where the ovarian stimulant was confirmed by examination of the records were included in two separate databases where the gonadotrophin was either purified, urinary FSH (Metrodin-HP, Serono Laboratories UK: `MHP'), or rFSH (Gonal-F, Serono Laboratories, UK: `GOF'). The MHP cycles were treated between January and December 1996, and the GOF cycles were treated between February 1997 and February 1998. The LH status of each cycle was determined in a plasma sample taken in the mid-follicular phase: stimulation day 7 (S7), when the first ovarian ultrasound scan was effected. The samples were stored for batch analyses of oestradiol and LH.
Hormone assays were performed using a semi-automated fluoroimmunometric system (Delfia; Wallac, Milton Keynes, UK) for LH in both databases, and oestradiol in the MHP database, while oestradiol was estimated using the Immulite chemiluminescent immunoassay system (Diagnostic Products Corporation, Caernafon, UK) in the rFSH database.
The circulating LH concentration used to identify those cycles with profoundly suppressed LH was taken as 0.7 IU/l (Fleming et al., 1998
), as the distribution of values indicated that this value represented the lowest quartile (Fleming, 1999
).
Retrospective analyses of the treatment databases
The functions of ovarian stimulation cycles that were deemed likely to be influenced by the circulating LH were examined for both databases. These were: the oestradiol concentrations after a fixed period of stimulation (on S7), and on the day of HCG administration; the `poor response' cancellation rates, the proportion of cases where the gonadotrophin dose was changed, and also the duration of stimulation. The other factors examined were oocyte yields, fertilization rates, embryo yields, the proportions of cases with embryo cryopreservation and finally the rates of pregnancy loss. These factors were examined for each of the complete treatment databases. Furthermore, all first treatment cycles were examined in isolation, as they represented an unbiased cohort and had a common starting dose (universally 225 IU FSH per day, for at least 6 days) and response assessment.
Statistical analyses
Due to changes in practice, and the non-randomized treatment schedules, the data could only be compared within databases based upon the discriminating LH value. Hormone concentrations were compared using t-tests, and group comparisons were compared using contingency table analyses. Data were deemed to be significantly different when P 0.05, and `marginally' significant when P
0.08 and P > 0.05.
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Results |
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Discussion |
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The effect of a reduced yield of oocytes in the MHP < LH cases was reflected in the lower proportion of cycles in the MHP < LH group with embryo cryopreservation. This clearly impacts upon the pregnancy potential of a treatment cycle. But again, there was no such effect in cycles treated with rFSH. To some degree the local treatment protocol influenced these results, since embryo cryopreservation was only affected when there was more than one quality of embryo available (data not shown). Thus the relatively small effect upon oocyte yield had a more profound effect upon embryo cryopreservation in the MHP < LH group. Although there was no effect of the LH suppression upon embryo scores (data not shown) the frequency of sufficient embryos of sufficient quality was critical to the cryopreservation data.
Ovarian hyperstimulation syndrome is a suitable candidate for analyses in database examinations such as these, especially as effects upon oestradiol concentrations have been observed, but the data were deemed unsuitable for comparisons within this context. This is because of the intrinsic bias incurred by the unit policy to cryopreserve all embryos when excessive numbers are retrieved, and also patients often refer themselves to other hospitals when in distress, such that reliable diagnoses and recording are not available.
The carbohydrate composition of rFSH, and consequently the isoform profile of rFSH, is different from that of highly purified FSH, and studies in vivo (Out et al., 1995) and in vitro (De Leeuw et al., 1996
) have suggested that rFSH is more potent than urinary-derived FSH in this clinical situation. In the data presented above, the oestradiol responses to stimulation were affected equally by the LH designation in both treatment databases, reflecting the direct role of LH in oestradiol biosynthesis. However, with rFSH treatment, suppression of LH failed to show the impact upon gross ovarian responses that were evident with purified urinary FSH (MHP).
Both FSH and LH induce increases in intracellular cyclic AMP (cAMP) activity in unluteinized follicular granulosa cells, and both are capable of stimulating aromatase activity. In fact, in mature granulosa cells, LH is more potent in the role of stimulating the cAMP internal messenger (Yong et al., 1994). Thus, low amounts of LH activity may contribute to the overall potency of an FSH preparation, as well as helping provide more androgen precursor for oestradiol biosynthesis. Therefore it is possible that differences in the effects of profound suppression of LH on ovarian responses in the two treatment schedules may be explained by differences in potency alone. The urinary FSH (MHP) may be less potent than the rFSH, but the low concentrations of LH remaining in the circulation may compensate for the `shortfall', while the rFSH is sufficiently potent to obviate the need for LH, except in its function of providing precursors for oestradiol biosynthesis.
Pregnancy rates with different drug treatment regimes have been addressed in other studies, with larger scope and formal randomization, with the general indication that treatment with FSH alone does not reduce pregnancy rates (Out et al., 1995) in IVF programmes where rFSH is used as the ovarian stimulant. However, pregnancy loss has not been explored with respect to suppressed follicular phase LH. Despite much interest in the role of excessive LH in early pregnancy loss (Homburg et al., 1988
), there has been no investigation into the effects of profound LH suppression into the viability of pregnancies achieved under these controlled conditions. If excessive activation of the LH receptor has implications for pregnancy viability, then it is possible that insufficient activation may be damaging also.
It has already been reported that the implantation potential of cryopreserved embryos was not affected by profound follicular phase LH suppression (Rehka et al., 1998) and furthermore that fresh blastocyst development in vitro was also unaffected (Fleming et al., 1998
). Pregnancy survival also appears to be unaffected by LH suppression, at least in the results from these two datasets (Table V
).
In conclusion, the data demonstrate that the impact of profound LH suppression depends upon the origin of the FSH, and that clinical implications may only apply when the ovarian stimulant is purified urinary FSH. In women with hypogonadotrophic hypogonadism, LH can be seen to sensitize follicles to FSH for recruitment and growth, as well as oestradiol secretion (ERHLHSG, 1998). However, although the degree of LH suppression achieved in ovarian stimulation cycles does not normally reach that of true hypogonadotrophic hypogonadism, similar effects based on potency can be detected with purified urinary-derived FSH. Subsequent potential effects of LH suppression upon oocyte fertilization rates, embryo developmental capacity and pregnancy survival were not identified in these analyses.
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Notes |
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References |
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Submitted on October 15, 1999; accepted on March 30, 2000.