1 MAR&Gen, Molecular Assisted Reproduction and Genetics, Gracia 36, 18002 Granada, Spain, 2 Laboratoire d'Eylau, 55 rue Saint-Didier, 75116 Paris, 3 ARCEFAR, 15 rue Faraday, 75017 Paris, France and 4 University of Granada, Campus Fuentenueva, 18004 Granada, Spain
5 To whom correspondence should be addressed. Email: cmendoza{at}ugr.es
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Abstract |
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Key words: ovarian stimulation/growth hormone/ovarian ageing/delivery rate/birth rate
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Introduction |
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Several studies have suggested that chromosomal abnormalities, mainly arising during the first meiotic division (Angell et al., 1993; Angell, 1997
; Dailey et al., 1996
), are essentially responsible for the age-related decline in oocyte developmental potential (Hassold et al., 1984
; Hassold and Chiu, 1985
; Kornafel and Sauer, 1994
). Interestingly, an impairment of meiotic spindle assembly has been reported in oocytes of older (4045 year old) women (Battaglia et al., 1996
). It is thus possible that the age-related decline of oocyte developmental potential may be at least partly caused by oocyte cytoplasmic abnormalities during final phases of intrafollicular development which, in their turn, may be related to an abnormal content of essential follicular fluid components.
We have shown previously that the ability of human oocytes to form morphologically normal and implantation-competent embryos is related to the concentration of different hormones in follicular fluid (Mendoza et al., 1999, 2002
). Among the hormones studied, growth hormone (GH) showed the most consistent relationship with different parameters of embryo quality, and higher concentrations of GH in follicular fluid were associated with rapid cleavage, good cleaving embryo morphology and a high embryo implantation potential (Mendoza et al., 1999
, 2002
). In a preliminary study (unpublished) we noted a decrease in follicular fluid GH concentration in women aged >40 years as compared with young women. The question then arises whether exogenous GH administration during ovarian stimulation can counteract the age-related decline of oocyte quality.
Exogenous GH has been shown to be beneficial in bovine superovulation protocols where it decreases the proportion of unfertilized oocytes and degenerate embryos (Cushman et al., 2001; Moreira et al., 2002
), although another recent study failed to confirm these observations (Hasler et al., 2003
). A beneficial effect of exogenous GH on the number of transferable embryos and birth rate was also described in superovulated ewes (Folch et al., 2001
).
Studies evaluating the benefits of co-treatment with GH during controlled ovarian stimulation for human assisted reproduction treatment have reported controversial findings. A randomized trial showed an improvement of ovarian response to gonadotrophins by GH co-treatment in women with polycystic ovaries (Owen et al., 1991), but another randomized study failed to confirm this effect (Homburg et al., 1995
). In patients with low pituitary GH reserve (clonidine-negative), but not in those with normal pituitary GH reserve (clonidine-positive), GH co-treatment reduced gonadotrophin requirement and increased pregnancy rate when combined with hMG for ovarian stimulation (Blumenfeld et al., 1994
). In another small series of 12 patients, a better fertilization and pregnancy rate was reported with GH co-treatment as compared to a previous attempt without GH (Wu et al., 1996
). An enhancement of in vitro maturation and fertilization of human germinal vesicle oocytes retrieved from small antral follicles by in vivo administration of GH has also been described (Hassan et al., 2001
). On the other hand, no effect of GH on the number of oocytes, embryos and pregnancies was obtained in two small series (seven and 22 patients respectively) of women with a poor ovarian response to gonadotrophins (Levron et al., 1993
; Suikkari et al., 1996
). A larger double-blind prospective study (21 patients in both the GH treatment and placebo arms) failed to show significant effects of GH on ovarian stimulation cycle characteristics and the number of oocytes collected, but pregnancy rate has not been evaluated (Hughes et al., 1994
).
It has to be remembered that both the positive and the negative data concerning the use of GH in ovarian stimulation were generated in small studies. Moreover, the target patient population was not always defined clearly, and the outcome measures did not always include delivery and live birth rates which are the most relevant parameters of assisted reproduction treatment success. In a recently updated Cochrane review including all randomized controlled trials employing GH in the ovarian stimulation, no effect of GH was noted in normal responders, but a significant improvement in live birth rate was found in poor responders, although this result was only just significant (Harper et al., 2003).
In this study we tested the usefulness of GH co-stimulation in a group of patients defined by advanced female age (>40 years). Delivery and live birth rate were used as the main outcome measures.
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Materials and methods |
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To compare assisted reproduction outcomes in women co-stimulated with GH with those in a placebo group, delivery rate (percentage of women under treatment who delivered a living newborn) and live birth rate (percentage of embryos transferred that gave rise to a living newborn) were chosen as the main outcome measures. In our previous series of attempts (non-prospective, non-randomized) the delivery rate in women of >40 years of age was 5%, and it reached 25% after co-stimulation with GH. It was calculated that some 50 attempts were needed to be performed in both the treatment and the control group to detect a difference of 20% (from 5 to 25%) with 80% power and 5% significance level (two-tailed test with alpha=0.05).
Controlled ovarian stimulation
All patients included in this study adhered to the same protocol consisting of pituitary down-regulation with triptorelin (Decapeptyl; Ipsen, Slough, UK) started in the mid-luteal phase at a dose of 0.1 mg/day. The dose was reduced to 0.05 mg/day from the day of the subsequent vaginal bleed. This reduced daily dose was administered until the day of ovulation-inducing hCG injection.
The starting dose of gonadotrophins was 450 IU of recombinant human FSH (rFSH, Puregon; Organon, Oss, The Netherlands) and 150 IU of hMG (Menopur; Ferring, Geneva, Switzerland), and this treatment was continued during the first 4 days of stimulation. From day 5 of stimulation the dose of gonadotrophins was adapted according to changes in the number and size of ovarian follicles and serum concentrations of estradiol and LH. These determinations were performed on day 5 and then every other day until the day of ovulation-triggering injection of hCG. According to the study design (see previous section), daily injections of 8 IU of GH or placebo were administered from day 7 of stimulation till the day following hCG administration. The chosen dose of 8 IU of GH daily was determined as an intermediate between the lowest dose (4 IU daily; Suikkari et al., 1996) and the highest dose (12 IU daily; Hughes et al., 1994
) reported in the literature. When at least one ovarian follicle had reached a diameter of 18 mm, ovulation was induced with 250 µg recombinant hCG (Ovitrelle; Serono, Geneva, Switzerland).
Assisted reproduction techniques
Oocyte recovery was performed by ultrasound-guided follicle aspiration under general anaesthesia. Oocytecumulus complexes were incubated in Gamete medium (Vitrolife, Göteborg, Sweden) for 24 h. The cumulus oophorus and the corona radiata were subsequently removed from the oocytes by a brief incubation in hyaluronidase solution (Hyase, Vitrolife) followed by repeated aspiration with plastic stripper tips (Mid Atlantic Diagnostics, Marlton, New Jersey, USA). After this procedure oocyte maturity was assessed, and all mature (metaphase II) oocytes were inseminated by ICSI which was performed within 3060 min after the cumulus oophorus and corona radiata removal. ICSI was carried out with the previously described technique and instruments (Tesarik and Mendoza, 2002).
After ICSI the oocytes were cultured at 37°C in IVF medium (Vitrolife) equilibrated with 5% CO2 in air for 1416 h. After this period, fertilization results were assessed. Normally fertilized oocytes (two pronuclei and two polar bodies) were separated and incubated in fresh medium of the same composition for an additional 2226 h when they were put to fresh culture medium again. All cultures were performed in 4-well culture dishes (Nunc, Roskilde, Denmark). All embryos (17) of each couple were grouped together in a single well containing 0.5 ml medium. Three days after ICSI, all living embryos developing from normally fertilized oocytes (15) were transferred to the patient's uterine cavity.
Hormone assays
Hormone assays used in this study were applied to both blood serum (estradiol, LH and GH) and follicular fluid (estradiol and GH) samples. Estradiol and LH were determined by using commercial enzyme immunoassay kits (Boehringer Mannheim, Mannheim, Germany). The intra- and inter-assay variabilities were 2.7 and 5.0% for estradiol, and 1.8 and 5.1% for LH. Direct radioimmunoassay was used for the determination of GH (Sorin Biomedica, Vercelli, Italy). The intra-and inter-assay variability for GH determination was 1.9 and 4.5% respectively.
Zygote and cleaving embryo evaluation
Zygotes were evaluated according to a previously described scoring system (Tesarik and Greco, 1999) simplified by grouping together all abnormal patterns (Tesarik et al., 2000
). Cleaving embryos were evaluated twice, first at the time of medium change 2 days after ICSI, and second 1030 min before transfer to the uterus 3 days after ICSI. Cleavage speed and embryo morphology were evaluated as described (Tesarik and Greco, 1999
). Embryos that had >3 cells 2 days after ICSI and >6 cells 3 days after ICSI and that showed <20% of space occupied by cell fragments were considered to have good morphology.
Statistical analysis
Differences between groups were assessed by two-tailed 2-test with Yates' correction or Fisher's exact test for categorical variables, and by MannWhitney U-test for continuous variables. All analyses were performed using the Statistica 5.0 package (Statsoft Version 5.1, Hamburg, Germany).
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Results |
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Discussion |
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It has to be noted that GH concentration in follicular fluid aspirated from women in the placebo group was low. Similar low GH concentrations in follicular fluid have previously been shown to be associated with assisted reproduction treatment failure (Mendoza et al., 2002). In contrast, in the GH co-treatment group the concentration of GH in follicular fluid was markedly higher. Taking into consideration the recent findings showing a requirement for GH to support ovarian follicular development in rat and mouse (Bachelot et al., 2002
; Zhao et al., 2002
) and the impact of GH insufficiency on ovarian function in women (Spiliotis, 2003
), the improvement of delivery and live birth rate by GH co-stimulation observed in the present study may be related to the correction of an age-related decrease in endogenous intrafollicular GH concentration during the final phase of oocyte meiotic maturation. Interestingly, a previous randomized, double-blind and placebo-controlled study reported an increase in follicular fluid GH and in pregnancy rate in low responders co-treated with pyridostigmine, an acetylcholinesterase inhibitor, during controlled ovarian stimulation (Kim et al., 1999
).
As to the secondary outcome measures, this study did not find any significant difference in the duration of ovarian stimulation, the total dose of gonadotrophins used, and the number of total and metaphase II oocytes recovered. These observations are in agreement with the conclusions of a Cochrane review compiling data of six small studies (Kotarba et al., 2000), but they differ from those of a European and Australian multicentre study (1995) in which GH co-stimulation led to a reduction of the gonadotrophin dose and shortening of the ovarian stimulation duration (European and Australian Multicenter Study, 1995
). Here again, this difference can be explained by the different character of the patient populations enrolled in both studies, namely women suffering from hypogonadotrophic hypogonadism (European and Australian Multicenter Study, 1995
) and normogonadotrophic or slightly hypergonadotrophic women with advanced age (this study).
On the other hand, peak serum estradiol concentration achieved during ovarian stimulation was higher in women co-stimulated with GH as compared with controls in this study. With the same numbers of large antral follicles and oocytes recovered by follicular aspiration in both groups, this observation means that there was more estradiol per large antral follicle in the GH co-treatment group. In fact, follicular fluid aspirates from women co-stimulated with GH contained more estradiol than those from controls. Since higher concentrations of estradiol in pre-ovulatory follicular fluid predict a higher chance of pregnancy (Mendoza et al., 2002), this observation suggests that the effect of GH on intrafollicular estradiol production may mediate the beneficial effect of GH on oocyte quality. Stimulating action of GH on the production of estradiol by ovarian follicular cells has previously been described (Mason et al., 1990
; Lanzone et al., 1996
). Further study is needed to determine whether effects of GH on intrafollicular estradiol concentration is the only mechanism involved in the improvement of oocyte quality in women of >40 years or whether other mechanisms also come into play.
Unlike sheep (Folch et al., 2001) and cattle (Moreira et al., 2002
), where co-stimulation with GH was reported to improve fertilization and preimplantation embryo development, we have not found any difference in fertilization rate and embryo morphology between the GH co-stimulation and the control group. Even though a decline in oocyte quality is known to be the principal cause of assisted reproduction treatment failure in ageing females (Schramm et al., 2002
), the contribution of GH effects on uterine receptivity, similar to those suggested by a study using GH in cattle reproduction (Moreira et al., 2003), cannot be excluded.
In conclusion, this prospective randomized study shows that women aged >40 years undergoing assisted reproduction treatment and co-stimulated with GH achieve more ongoing pregnancies and suffer less pregnancy wastage, resulting in more deliveries and live births, as compared with women of the same age category stimulated with gonadotrophins alone. Further study is needed to determine the mechanism of this effect.
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Submitted on February 18, 2005; resubmitted on March 28, 2005; accepted on April 11, 2005.
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