1 MAR&Gen, Molecular Assisted Reproduction and Genetics, Gracia 36, 18002 Granada, and 2 Department of Biochemistry and Molecular Biology, University of Granada, Granada, Spain
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Abstract |
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Key words: LH/oocyte developmental competence/oocyte donation/ovarian stimulation/pituitary down-regulation
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Introduction |
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The actual importance of LH during ovarian stimulation is a matter of debate. Some authors have reported lower estradiol (E2) biosynthesis (Fleming et al., 1996; Westergaard et al., 1996
; De Placido et al., 2001
), lower oocyte and embryo yield (Fleming et al., 2000
; De Placido et al., 2001
) and a higher frequency of early pregnancy wastage (Westergaard et al., 2000
) in normogonadotrophic women down-regulated with a GnRH agonist and stimulated with highly pure FSH preparations when compared with women stimulated with hMG or with a combination of hMG and FSH. However, other authors failed to detect any relationship between serum LH levels and ovarian stimulation outcomes (Balasch et al., 2001
). Reflecting this conceptual confusion, some authors advocate the addition of hormone preparations containing LH activity to ovarian stimulation protocols if serum LH activity falls below a certain threshold level (Filicori et al., 1999
, 2001
), whereas others do not recommend any additional exogenous LH supplementation (Balasch et al., 2001
).
There may be several reasons for these discrepancies. First, individual studies differ as to the definition of the threshold serum LH level below which exogenous LH supplementation is considered. Further, LH may affect IVF results both by determining oocyte quality and by influencing uterine receptivity via ovarian secretion of E2 or through direct effects on endometrium, myometrium, and uterine artery and vein (Rao, 2001; Shemesh, 2001
).
This prospective, randomized study was designed to address the question of LH effect on oocyte yield and quality, independently of extragonadal LH actions. To this goal, this study was performed in a group of young, normogonadotrophic oocyte donors. Embryos resulting from the donated oocytes were transferred to patients, all of whom received the same treatment to prepare the uterus for implantation, thus avoiding the possible influence of LH on uterine receptivity. Within the group of oocyte donors, the effects of exogenously administered LH are related to the degree of endogenous LH suppression.
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Materials and methods |
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The age of the male patients ranged between 2862 years, and that of the female patients ranged between 3350 years. Each oocyte donor was involved only once in this study, and the oocytes obtained from each donor were shared between two infertile couples. The choice of oocyte donor for each infertile couple was made merely on the basis of physical resemblance between the donor and the patient. Accordingly, donor oocytes were distributed among different couples at random with regard to male and female age and the status of the reproductive function.
The indications for oocyte donation were premature or physiological menopause without spontaneous cycle (n = 302), pre-menopausal with poor ovarian response in previous ovarian stimulation attempts (n = 198) and repeated unexplained failures of assisted reproduction attempts that were believed to be of oocyte origin (n = 6). Out of the 506 male partners of the female patients involved in this study, 290 were normozoospermic according to the World Health Organization criteria (World Health Organization, 1992). Spermogram and spermocytogram values were moderately impaired in 156 and severely impaired in 60 of these men. Cases of azoospermia, requiring the recourse to testicular biopsy to obtain sperm, are not included in this study.
Study design and ovarian stimulation
All patients receiving embryos resulting from donated oocytes were prepared in the same way (see below). Oocyte donors were down-regulated with triptorelin (Decapeptyl 3.75 mg; Ipsen Pharma, Barcelona, Spain) administered on cycle day 24. They received oral norethisterone (Primolut Nor; Shering, Madrid, Spain), 10 mg/day between cycle days 21 and 27 to reduce the risk of ovarian cyst formation in response to triptorelin. After menstrual bleeding the achievement of pituitary down-regulation was assessed by determination of serum E2 concentration (<45 pg/ml) and by vaginal ultrasonography (absence of follicles and cysts of >10 mm in diameter). The donors were kept in the down-regulated state until the down-regulation of the respective oocyte recipients was achieved. At that time ovarian stimulation was started. The time between the menstrual bleeding following triptorelin administration and the beginning of ovarian stimulation ranged between 2 and 12 days.
One to three days before the beginning of ovarian stimulation, serum LH level was determined with the use of a commercial enzyme immunoassay kit (Vidas LH; BioMérieux, Lyon, France). The detection limit and the inter-assay coefficient of variation were 0.05 IU/l and 3.2%, respectively. The intra-assay coefficient of variation was <4.5% in the range of LH concentrations measured in this study. Oocyte donors with LH <1 IU/l were allocated at random to two groups (group I or group II) using a computer-generated randomization list. Donors allocated to group I were stimulated with FSH alone, whereas those allocated to group II received additional LH during the stimulation period. Donors with LH 1 IU/l were also randomized to two groups to be stimulated with FSH alone (group III) or a combination of FSH and LH (group IV).
During the first 4 days of ovarian stimulation, all oocyte donors received the same treatment. It consisted of s.c. administration of 150 IU rFSH (Puregon; Organon, Oss, The Netherlands) on days 1 and 2 of stimulation and 100 IU rFSH (Puregon) on days 3 and 4 of stimulation. On day 5 of stimulation, serum E2 and LH levels were determined, and the number and size of ovarian follicles were evaluated by vaginal ultrasonography. The daily injections of FSH during the whole period of ovarian stimulation were administered between 18.00 h and 20.00 h, whereas blood samples for determination of E2 and LH were taken between 9.00 h and 11.00 h on the corresponding days of stimulation.
On day 5, the stimulation protocol was adapted as follows (Figure 1). In group I ovarian stimulation was continued with FSH alone, whereas donors allocated to group II received exogenous LH, in addition to FSH, on days 57 of stimulation (Figure 1
). This treatment was realised by daily injection of 1 vial of hMG (Menogon; Langley, Berkshire, UK), containing 75 IU of FSH and 75 IU of LH activity, on these days. In addition, both group I and group II were subdivided on day 5 of stimulation according to serum E2 concentration (Figure 1
). If serum E2 was <100 pg/ml (groups Ia and IIa), the daily dose of FSH was increased by adding 75 IU of urinary FSH (uFSH) on days 57. In group IIa this additional FSH was contained in the hMG preparation (Menogon) added on these days, whereas the corresponding dose increase in group Ia was realised by adding 75 IU of purified uFSH (Neofertinorm; Serono, Rome, Italy). If serum E2 on day 5 of stimulation was
100 pg/ml (groups Ib and IIb), the dose of rFSH was lowered to 50 IU/day, while 75 IU uFSH was added in the form of Neofertinorm (Serono) or Menogon (Ferring) in groups Ib and IIb respectively (Figure 1
).
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Beginning with day 8 of stimulation, the dose of rFSH administered to oocyte donors became group-independent (Figures 1 and 2) and was adapted to the individual increase in serum E2 level and the number and size of ovarian follicles determined by vaginal ultrasonography. Both measurements were performed every other day. In addition, donors in groups I and III continued to receive the daily dose of 75 IU of uFSH (Neofertinorm), while donors in groups II and IV received 1 vial of Menogon (75 IU of uFSH and 75 IU of LH).
Ovarian puncture was performed on day 14 of stimulation, in phase with the protocol of the corresponding oocyte recipient preparation (see below). Ovulation was induced by i.m. injection of 10 000 IU of HCG (Profasi, Serono, Rome, Italy) 36 h before ovarian puncture. All donors involved in this study had 5 follicles of >18 mm in diameter on the day of HCG administration.
Oocyte recipient preparation
Patients who still had spontaneous menstrual bleeding were first subjected to pituitary down-regulation with a long-acting GnRH agonist preparation (triptorelin; Decapeptyl 3.75 mg; Ipsen Pharma), administered between days 2128 of the cycle. The day of triptorelin administration was determined taking into account the date of the patient's last menstrual bleeding, the planned date of embryo transfer and the usual duration of the patient's menstrual cycle. To reduce the risk of ovarian cyst formation in response to the GnRH agonist, patients either took a contraceptive pill during the cycle in which GnRH agonist was administered or were given oral norethisterone (Primolut Nor), 10 mg daily, for 7 days beginning 3 days before, and ending 3 days after, the application of GnRH agonist. Patients who lacked spontaneous menstrual bleeding were given a contraceptive pill (Ovanon; Organon, Puteaux, France) for at least one cycle prior to commencing endometrial preparation for embryo transfer.
Endometrium growth was stimulated by progressively increasing daily doses of orally administered estradiol valerate (Progynova; Schering) followed by vaginally administered natural micronized progesterone (Utrogestan; Laboratoires Besins-Iscovesco, Paris, France). Details of these treatments have been published earlier (Tesarik et al., 2002). The time between the administration of GnRH agonist and the beginning of endometrial growth stimulation ranged between 12 and 26 days.
Assisted reproduction techniques
Oocyte-cumulus complexes were incubated for 24 h in IVF-50 medium (Vitrolife; Göteborg, Sweden) equilibrated with 5% CO2 in air before performing ICSI. Shortly before ICSI the cumulus oophorus and the corona radiata were removed from the oocytes by incubation with 20 IU/l hyaluronidase (Hyase; Vitrolife) solution prepared with equilibrated Gamete 100 medium (Vitrolife). The incubation was carried out at 37°C for 2030 s.
Within 1 h following the cumulus oophorus and corona radiata removal, oocytes were subjected to ICSI using previously described techniques and instruments (Tesarik and Sousa, 1995). Fertilization outcomes were evaluated by a single observation of the oocytes 1516 h after ICSI. Oocytes were considered to be normally fertilized when they showed 2 pronuclei (PN) and 2 polar bodies (PB) at that time. The numbers of abnormally fertilized/activated oocytes (1 PN and 2 PB or >2 PN with 1 or 2 PB) and of oocytes that failed to be activated (metaphase II) were also noted.
Normally fertilized oocytes were incubated further at 37°C in IVF-50 medium (Vitrolife) equilibrated with 5% CO2 in air as described (Tesarik et al., 2002). On day 3, 24 embryos with the best morphological grade (see below) were selected and transferred to the patient's uterus. Only embryos graded as excellent or good were transferred in all treatment attempts. The remaining embryos were cryopreserved. This study only involves data obtained with fresh embryo transfers.
After embryo transfer, the patients were given oral estradiol valerate (Progynova; 4 mg/day), aspirin (Bayer, Madrid, Spain, 100 mg/day), and vaginally administered natural micronized progesterone (Utrogestan; Laboratoires Besins-Iscovesco, 600 mg/day). Pregnancy was assessed 12 days after embryo transfer by determining serum ß-HCG concentration. In case of pregnancy the treatment with estradiol valerate and aspirin was continued up to the end of the third week after embryo transfer, and the treatment with progesterone was prolonged until the end of the third month after transfer.
Pronuclear zygote and cleaving embryo grading
Normally fertilized, two-pronucleated zygotes were evaluated within an interval of 1516 h after ICSI using previously described scoring criteria (Tesarik and Greco, 1999). The scoring system was simplified by grouping all abnormal pronuclear patterns into a single group as described earlier (Tesarik et al., 2000
).
Cleaving embryos were graded on day 3 after ICSI using criteria based on the cleavage speed and morphology (blastomere regularity and the volume occupied by anucleate cell fragments). The latter was quantified with the used of previously described embryo morphology-grading criteria (Bolton et al., 1989). The original grades 14 are presented as excellent, good, fair and poor respectively.
Statistics
Means were compared by ANOVA and paired Student's t-test. Proportions were compared by 2-test. All statistics were performed using StatView II statistical package (Abacus Concepts, Berkeley, California, USA). Unless stated otherwise, values are considered as significantly different if P < 0.05.
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Results |
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The peak serum E2 concentration achieved in the course of ovarian stimulation tended to be higher in all groups in which LH was added to the ovarian stimulation protocol, but the difference from the corresponding group stimulated with FSH alone was significant only in donors with low serum LH before the beginning of stimulation and low E2 rise on day 5 of stimulation (Table II). Total FSH dose per stimulated cycle was significantly lower in all groups in which LH was added to the stimulation protocol when compared with corresponding groups of donors stimulated with FSH alone (Table II
).
Oocyte yield and maturity
The number of mature (metaphase II) oocytes per donor was higher in all groups co-stimulated with LH when compared with corresponding groups stimulated with FSH alone (Table III). The number of immature (germinal vesicles and metaphase I) oocytes was similar in all groups except for groups IVa and IVb, formed by donors with initial LH
1 IU/l and receiving exogenous LH as part of the stimulation protocol, in which significantly more immature oocytes were recovered (Table III
).
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Zygote and embryo morphology
In the group of donors with low pre-stimulation serum LH, the addition of LH to the ovarian stimulation protocol increased both the number of good-morphology zygotes on day 1 following ICSI and the numbers of cleaving embryos graded as excellent or good on day 3 after ICSI when compared with corresponding groups of donors stimulated with FSH alone (Table IV). This increase was mainly related to the higher number of normally fertilized oocytes in the groups co-stimulated with LH, because the number of poor-morphology zygotes and that of embryos graded as fair and poor was also slightly but significantly augmented in these groups when compared with corresponding groups of donors stimulated with FSH alone (Table IV
). In contrast, no benefit from exogenous LH addition, in terms of the yield of good-morphology zygotes and embryos, was observed in those groups formed by donors with pre-stimulation serum LH of
1 IU/ml (Table IV
). On the contrary, the number of poor-morphology zygotes was higher and the number of excellent-morphology cleaving embryos was lower in the groups of donors with pre-stimulation serum LH of
1 IU/1 and co-stimulated with exogenous LH when compared with groups of donors with similar characteristics stimulated with FSH alone (Table IV
).
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Discussion |
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A clue to the understanding of this apparent discrepancy can be found in the dynamics of follicular recruitment during stimulation. According to the protocol of this study, all donors received the same treatment during the first 4 days of stimulation. Hence, day 5 cycle characteristics reflected the individual variability of the ovarian response. On day 8, when consequences of the differences in the ovarian stimulation protocol in different groups could be appreciated, donors with initially low serum LH and receiving exogenous LH had higher serum E2 concentration and more follicles of >8 mm in diameter when compared with those stimulated with FSH alone. Because more metaphase II oocytes, normally fertilized oocytes, good-morphology zygotes and embryos and implanted embryos were obtained in those donors with pre-stimulation serum LH of <1 IU/l who were co-stimulated with exogenous LH, it is evident that some of the follicles recruited between days 58 were still capable of yielding developmentally competent oocytes.
In contrast, follicles newly recruited between days 58 in donors with pre-stimulation serum LH level of 1 IU/l and co-stimulated with exogenous LH were mostly developmentally incompetent because the numbers of mature oocytes, normally fertilized oocytes, good-morphology zygotes and embryos, and the implantation rate were not increased when compared with donors stimulated with FSH alone.
In the groups of donors with low pre-stimulation serum LH levels, serum LH showed a further decrease during the first 5 days of stimulation. Under these conditions, some healthy small antral follicles may fail to be recruited; such follicles may thus be `rescued' by the subsequent addition of exogenous LH, although this exogeneous LH contribution was not reflected by a measurable increase in serum LH. Rescue of IVF cycles in pituitary down-regulated normogonadotrophic young women with a poor initial response to rFSH has been reported (De Placido et al., 2001. These data are similar to the observations of this study concerning oocyte donors with a low pre-stimulation serum FSH level. In a previous randomized comparative study dealing with the effects of highly purified FSH versus hMG in an oocyte donation programme (Söderström-Anttila et al., 1996), using a similar pituitary down-regulation protocol as that applied in the present study, oocytes from donors stimulated with hMG also showed a higher fertilization rate when compared with oocytes from donors stimulated with FSH alone. In our study we could not confirm this observation. This may be caused by the different technique of assisted reproduction used, namely ICSI in our study and conventional IVF in a previous study (Söderström-Antilla et al., 1996
).
In the groups of donors with pre-stimulation LH level of 1 IU/l, the addition of exogenous LH commencing on day 5 of stimulation also led to further follicular recruitment. However, these groups showed a higher degree of previous follicular recruitment during the first 5 days of stimulation, and this initial wave may have involved most, if not all, of the healthy follicles available in the present cycle. The late recruitment between days 58 of stimulation may thus mainly concern developmentally incompetent follicles in these donor groups. This would explain why the addition of exogenous LH in donors with initially high serum LH level did not improve the yield of developmentally competent oocytes and embryos.
The addition of exogenous LH in donors with initially high LH levels impaired the oocyte and embryo quality and decreased the implantation rate, although the latter was not significant. This latter observation cannot be explained by the late recruitment of developmentally incompetent follicles, and some of the originally recruited healthy follicles are likely to have deteriorated during subsequent ovarian stimulation of these donors to a higher degree than follicles in the corresponding groups of donors stimulated with FSH alone. It is possible that the strong stimulation of theca interna cells by the conjoint action of endogenous and exogenous LH in the mid-follicular phase of these donors may have led to excess androgen production in some follicles, whose granulosa cell compartment did not produce sufficient aromatase activity to efficiently convert this excess androgen to estrogen. This might lead to a shift of the intrafollicular androgen-to-estrogen ratio in favour of androgen. Under in-vitro conditions, estradiol has been shown to improve cytoplasmic maturation and early post-fertilization development of human oocytes (Tesarik and Mendoza, 1995), whereas androstenedione irreversibly blocked the estradiol effects (Tesarik and Mendoza, 1997
). It is possible that relative over-production of androgens in vivo may have a similar effect, although no direct experimental evidence for this has yet been provided.
These data support the concept of a `window' for LH requirement in ovarian follicular development, originally formulated by Hillier, according to which there is not only a threshold requirement for LH but also a ceiling level beyond which LH might be deleterious to ovarian stimulation (Hillier, 1994). It is noteworthy that the poor embryo quality was detected as early as the pronuclear zygote stage. Thus, similar to paternal effects (Tesarik et al., 2002
), maternal, oocyte-borne effects on human embryo development also became manifest as early as the first cell cycle after fertilization.
The present data, obtained in an oocyte donation programme, may help the understanding of some discrepancies between previously published studies of which some claimed (Filicori, 1999; Filicori et al., 1999
; 2001
; Fleming et al., 2000
; Westergaard et al., 2000
, 2001
; De Placido et al., 2001
; Meo et al., 2002
) and others negated (Balasch et al., 1996
, 2001
) effects of exogenous LH on ovarian stimulation outcomes. First of all, the combination of norethisterone and a depot GnRH agonist preparation, used in this study for pituitary down-regulation of oocyte donors, strongly suppresses endogenous LH levels when compared with protocols using a shorter treatment with short-acting GnRH agonist preparations only. Hence, the effect of exogenous LH supplementation may be more perceptible on this strongly suppressed background as compared with studies using milder down-regulation schemes. Furthermore, the effect of LH on oocyte quality is likely to be less perceptible in those groups of patients undergoing IVF with their own oocytes when compared with oocyte donation. For instance, strong response to ovarian stimulation, leading to the production of numerous good-quality oocytes, may be deleterious to uterine receptivity (Paulson et al., 1990
; Simon et al., 1998
). Finally, the relative paucity of embryos with high implantation potential, strongly perceived in an oocyte donation programme where embryos from a single donor are shared by several recipients, may escape attention in IVF with the patient's own oocytes where only a few embryos with the best quality are selected for transfer.
When only the impact on oocyte number and developmental competence is taken into account, this study suggests that exogenous LH during ovarian stimulation can be beneficial in some women and detrimental in others. The level of serum LH before the beginning of stimulation or on day 5 of stimulation may become a useful criterion to distinguish between these two conditions, but the optimal cut-off values still remain to be determined.
When extrapolating these findings to poor-responder management, the inclusion of exogenous LH can be expected to increase oocyte yield but should be used with caution to avoid the deterioration of oocyte quality. In good responders, the risk of the development of severe ovarian hyperstimulation syndrome and of deterioration of uterine receptivity by excessive serum E2 levels must also be taken into account.
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Notes |
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References |
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Submitted on April 10, 2002; resubmitted on June 12, 2002; accepted on August 12, 2002.