Department of Obstetrics & Gynaecology, Queen Mary Hospital, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region, People's Republic of China
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Abstract |
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Key words: HMG/ICSI/oocyte and embryo quality/ovarian stimulation/recombinant human FSH
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Introduction |
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Human menopausal gonadotrophin (HMG) containing 75 IU FSH and 75 IU LH is extracted from the urine of post-menopausal women and is of low purity as 95% of the proteins are contaminants. The urinary proteins may have negative effects on follicular recruitment and development (Giudice et al., 1994). Recombinant human FSH (rhFSH) is produced by a Chinese hamster ovary cell-line, transfected with the genes encoding for the two FSH sub-units. This results in an almost totally pure FSH preparation. Both HMG and rhFSH are effective in inducing follicular growth and maturation. Clinical studies have shown that rhFSH is an efficacious compound as compared to HMG (Recombinant Human FSH Study Group, 1995
) and urinary FSH (Out et al., 1995
; Bergh et al., 1997
). Daya et al. (1995) in a meta-analysis suggests that the use of FSH is associated with a better outcome than HMG (Daya et al., 1995
), although this is not supported by another recent meta-analysis (Agrawal et al., 2000).
Most of these trials focus on clinical parameters such as number of oocytes obtained, dosage/duration of gonadotrophin used, fertilization and pregnancy rates. Fertilization and pregnancy rates are not sensitive indicators of oocyte quality as they can be affected by other factors such as semen quality and uterine receptivity. There are, however, very few studies examining the effects of different gonadotrophin preparations on oocyte and embryo quality. Conflicting reports exist in the literature. Imthurn et al. (1996) found a significantly higher proportion of metaphase II oocytes and fewer oocytes with dark cytoplasm in women receiving highly purified urinary FSH , when compared with those receiving HMG (Imthurn et al., 1996). Similar findings have been shown (Mercan et al., 1997
). On the other hand, no such improvement in the nuclear maturity of oocytes was demonstrated when FSH was used (Jacob et al., 1998
; Weissman et al., 1999
).
The objective of this prospective, randomized study was to compare the effects of HMG and rhFSH on oocyte and embryo quality. There are still no published reports comparing HMG and rhFSH in this context. The hypothesis was that oocyte and embryo quality was significantly improved after using rhFSH, compared with HMG.
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Materials and methods |
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Patients had to fulfil the inclusion and exclusion criteria before they were recruited in this study. Inclusion criteria included: (i) age of women <40 years; (ii) day 2 serum FSH concentration <10 IU/l; (iii) regular ovulatory cycles; (iv) long protocol of pituitary down-regulation; (v) intracytoplasmic sperm injection (ICSI) indicated for severe male factors, i.e. <100 000 motile spermatozoa recovered after sperm preparation or surgical retrieval of spermatozoa from epididymis or testis in the case of obstructive azoospermia. Exclusion criteria were: (i) smokers; (ii) history of ovarian cystectomy or oophorectomy; (iii) short protocol of pituitary down-regulation; (iv) ICSI required for failed fertilization or low fertilization rates (<30%) in previous cycles; (v) testicular sperm extraction required. Every patient was extensively counselled and gave an informed consent prior to participating in the study, which was approved by the Ethics Committee, Faculty of Medicine, The University of Hong Kong.
Assignment
Patients were randomized on the first day of gonadotrophin injection according to a computer-generated list of random numbers, which were placed in the sealed envelopes. The list was generated by a nurse not involved in IVF treatment and the nurse working in the assisted reproduction unit would open the sealed envelopes according to the sequence.
Ovarian stimulation
The details of the long protocol of ovarian stimulation regimen used at our centre have been previously published (Ng et al., 2000). In short, women were all pre-treated with buserelin (Suprecur; Hoechst, Frankfurt, Germany) nasal spray 150 µg four times a day from the mid-luteal phase of the cycle preceding the treatment cycle. Pituitary down-regulation was confirmed by both transvaginal scanning and serum oestradiol performed on the second day of the treatment cycle. Concentrations of serum FSH and LH were also measured prior to stimulation. Patients received a standard protocol of either HMG (Pergonal; Serono, Geneva, Switzerland) i.m. or rhFSH (Gonal-F; Serono) s.c. In the first 2 days, 300 IU HMG or rhFSH were given, which was followed by 150 IU HMG or rhFSH daily. The ovarian response was monitored by serial transvaginal scanning and serum oestradiol concentrations. Human chorionic gonadotrophin (HCG, Profasi; Serono) 10 000 IU was given i.m. when the leading follicle reached 18 mm in diameter and there were at least three follicles >15 mm in diameter. Blood was taken on the day of HCG for serum oestradiol, FSH and LH concentrations. Transvaginal ultrasound-guided oocyte retrieval was scheduled 36 h after the HCG injection.
Masking
The embryologists (E.Y.L.L. and W.S.B.Y.) performing the assessment of oocytes and ICSI were blind to the type of gonadotrophin each patient had received for stimulation.
ICSI and assessment of oocyte and embryo quality
About 2 h after oocyte retrieval, the oocytes were denuded of their surrounding cumulus and corona radiata cells by using hyaluronidase (Sigma H4272; Sigma, St Louis, MO, USA) at 100 units/ml in HEPES buffered Earle's balanced salt solution (EBBS, Sigma E7883; Sigma) and aspirating oocytes through a fine capillary. Denuded oocytes were cultured in EBBS supplemented with 0.35% Plasmanate (Cutter 61325; Bayer Corporation, Elkhart, IN, USA) for 2 h. Prior to microinjection, each denuded oocyte was videotaped at two different planes for detailed assessment later, including (i) nuclear maturity classified into: germinal vesicle, metaphase I, metaphase II and atretic; (ii) morphology of zona pellucida, oocyte and polar body; and (iii) measurement of the zona thickness and diameters of oocyte and ooplasma. Oocytes were assessed according to Veeck (1999). A zona pellucida with septum, extension, uneven thickness or non-spherical shape was regarded to be abnormal. A normal oocyte had a clear homogeneous appearance throughout the whole cytoplasm. Any oocyte with area of saccules, vacuoles, refractile bodies, localized granularity or irregular shape was considered to be abnormal. A normal polar body was referred to a single distinct polar body.
ICSI was performed on metaphase II oocytes only. The injected oocyte was further cultured in a 15 µl droplet of EBSS medium under mineral oil in a CO2 incubator. Fertilization was checked 1418 h after the injection procedure. An oocyte was considered to be normally fertilized when two pronuclei were visible. When no pronucleus or only one pronucleus was visible, the oocytes were cultured for another 34 h and examined again. A maximum of three normally cleaving embryos were replaced into the uterine cavity 48 h after the retrieval. Excess good quality embryos were frozen for subsequent transfer if the patient did not conceive in that cycle. All fresh good quality embryos were cryopreserved if serum oestradiol on the day of HCG exceeded 30 000 pmol/l or features suggestive of ovarian hyperstimulation syndrome (OHSS) were present in order to reduce the risk of developing severe OHSS. Immediately before the transfer or cryopreservation, embryos were examined for the number/regularity of blastomeres and the degree of fragmentation. Embryos were graded according to the criteria of Veeck (Veeck, 1988).
Luteal phase was supported by 1500 IU HCG injections on the day of embryo transfer and 6 days later. A urine pregnancy test was done 16 days after embryo transfer. If it was positive, ultrasound examination was performed 1014 days later to confirm intrauterine pregnancy and to determine the number of gestation sacs present.
Statistical analysis
The fertilization rate was defined as the proportion of oocytes resulting in two pronuclei formation. Only clinical pregnancies were considered in this study, which were defined by the presence of one or more gestation sacs on scanning. The mean implantation rate was the proportion of embryos transferred resulting in an intrauterine gestational sac.
In the report of Imthurn et al. (1996), the proportions of metaphase II oocytes in the HMG group and the urinary FSH group were 80.6 and 88.8% respectively (Imthurn et al., 1996). On average, we obtained 11 oocytes per retrieval and 85% of the oocytes were of metaphase II. Assuming an increase of 10% in the nuclear maturity after using the rhFSH as significant, the number of metaphase II oocytes in each group should be 208 to give a test of significance of 0.05 and a power of 0.8 (Sigmastat, Jandel Scientific, San Rafael, CA, USA). Twenty subjects were required in each arm.
The primary outcome measure was the percentage of metaphase II oocytes. Other outcome measures included the percentage of normal zona/oocyte/polar body morphology, zona thickness, diameters of oocyte and ooplasma, blastomere number and grading of embryos. Statistical comparison were carried out by the Mann-Whitney U-test, Fisher's exact test, 2-test and Student's t-test, where appropriate. Two-tailed P < 0.05 was taken as significant.
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Results |
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Analysis
No significant differences were found between two groups with regard to the age of women, body mass index, the type and duration of infertility and the basal FSH level (Table I). Serum oestradiol, FSH and LH concentrations on the second day of the treatment cycle prior to stimulation were not different (data not shown). The ovarian responses, hormonal profile on the HCG day and treatment outcomes of two groups are summarized in Tables II and III.
Embryo transfer was not performed in four cycles of the HMG group and in three cycles of the rhFSH group because of the risk of developing OHSS. No significant differences were observed in the duration and dosage of HMG/rhFSH, the hormonal profile on the HCG day, the number of cumulus-oocyte complexes and metaphase II oocytes obtained, the fertilization rate and the number of embryos transferred/frozen between two groups. There was a trend in pregnancy and implantation rates in favour of HMG although the difference did not reach statistical significance. All pregnancies were ongoing and referred out for antenatal care at 1012 weeks gestation.
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Discussion |
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Daya et al. (1995) in a meta-analysis demonstrated that the use of urinary FSH or rhFSH was associated with a significantly higher clinical pregnancy rate than HMG (Daya et al., 1995). Very low-level or the absence of LH in the urinary FSH or rhFSH preparation was considered as the main reason to explain >50% improvement in clinical pregnancy rates in women receiving FSH. A more recent meta-analysis (Daya and Gunby, 1999
) further indicated that rhFSH was superior to urinary FSH in terms of the pregnancy rate. A higher bioactivity of rhFSH is convincingly shown by lower FSH dosage/duration and more oocytes retrieved in women receiving rhFSH, when compared with urinary FSH (Out et al., 1995
; Bergh et al., 1997
). More basic FSH isoforms in the rhFSH preparation and the presence of FSH-inhibiting substances in urinary FSH may also explain the higher effectiveness of rhFH (Out et al., 1996
). It was further postulated that rhFSH might have direct effects on oocyte and embryo quality, leading to higher implantation rates.
Imthurn et al. (1996) in a prospective study examined the nuclear maturity of oocytes in women undergoing ICSI and receiving urinary FSH (Metrodin HP; Serono) and HMG (Pergonal; Serono) in a short protocol of pituitary down-regulation. The urinary FSH group, compared with the HMG group, had a significantly higher proportion of metaphase II oocytes (88.8 versus 80.6% respectively) and morphologically normal oocytes (85.6 versus 77.6% respectively). This study suffered from the use of a short protocol associated with very high follicular phase LH concentrations, exclusion of cycles with fewer than three oocytes from the analysis and cross-over design in 10 subjects. In a retrospective analysis (Mercan et al., 1997) of conventional IVF cycles in a long protocol of down-regulation, urinary FSH (Metrodin; Serono) stimulation resulted in a significantly higher percentage of mature oocytes than the urinary FSH/HMG (Metrodin/Pergonal) combination (57 versus 34% respectively). The drawbacks of this study were the retrospective nature and examination of conventional IVF cycles. The removal of the corona-cumulus complex before the ICSI procedure allows a more precise determination of nuclear maturity of the oocyte (Rattanachaiyanont et al., 1999
).
Contrary to the above two studies, Jacob et al. (1998) retrospectively demonstrated a similar percentage of metaphase II oocytes in women receiving HMG (Humegon; Organon, Oss, The Netherlands) and rhFSH (Puregon; Organon) in down-regulated cycles for ICSI (Jacob et al., 1998). Similarly, another retrospective study (Weissman et al., 1999
) found a comparable percentage of metaphase II oocytes retrieved after a long protocol of pituitary down-regulation and ovarian stimulation with HMG (Humegon or Pergonal) or urinary FSH (Metrodin HP) in women undergoing ICSI. Because of the conflicting evidence, it remains uncertain whether the use of FSH (especially rhFSH) will increase the percentage of nuclear mature oocytes after ovarian stimulation, resulting in better oocyte and embryo quality.
Oocyte and embryo quality can be adversely affected by a number of factors, including advanced maternal age (Sherins et al., 1995), raised basal FSH concentration and smoking (Zenzes et al., 1997
). Smokers were excluded in this study and all women recruited were <40 years old and had basal FSH concentration <10 IU/l. The fertilization rate, cleavage rate and pregnancy rate were significantly lower in patients with previously failed fertilization in conventional IVF compared to those with male factors, probably related to oocyte defects (Gabrielsen et al., 1996
). Therefore, only ICSI cycles indicated for severe male factors were examined. The presence of endometriosis may lead to impaired embryo quality related to an abnormal follicular hormonal environment (Garrido et al., 2000
). Diagnostic laparoscopy was not indicated in patients with severe male factors but chocolate cysts were not observed on scanning prior to stimulation in all patients in this study.
The type of ovarian stimulation, timing of HCG injection, interval between HCG and oocyte retrieval can also affect the oocyte and embryo quality. The use of a short protocol may result in a significantly lower proportion of metaphase II oocytes than a long protocol (Greenblatt et al., 1995). All patients received a standard protocol of ovarian stimulation including a long protocol of pituitary down-regulation and the same starting dose of gonadotrophins. This is not a double-blind study as the clinicians were aware of the type of gonadotrophins the patients were receiving for ovarian stimulation. The criteria for administering HCG were, however, essentially the same for all patients. The follicular size and the developmental potential of an oocyte in the stimulated ovary are not closed related and can be independent (Salha et al., 1998
). All oocyte retrievals were scheduled 36 h after the HCG injection.
Oocyte quality may also be impaired in patients with severe OHSS (Aboulghar et al., 1997; Akagbosu et al., 1998
). Median oestradiol concentrations on the HCG day and the number of embryo transfer to be cancelled because of the risk of developing OHSS were similar in two groups. Patients who are at risk of severe OHSS usually have polycystic ovaries. It is still unclear whether the quality of oocytes and embryos is impaired in patients with polycystic ovaries (Hardy et al., 1995
; Aboulghar et al., 1997
; Ludwig et al., 1999
). All patients in this study had regular ovulatory cycles and did not have typical features of polycystic ovaries on scanning.
This is the first prospective and randomized study comparing the effects of rhFSH and HMG on the quality of oocytes and embryos. The embryologists performing the assessment were blind to the type of gonadotrophin each patient had received for stimulation. As HMG contains equal proportions of LH and FSH and is contaminated with >95% non-FSH urinary proteins, we anticipated marked differences in oocyte and embryo quality between the HMG and rhFSH groups. To our surprise, our results cannot demonstrate any significant differences between the HMG and rhFSH groups with regard to oocyte and embryo quality. Serum concentrations of LH on the HCG day were low (median ~2 IU/l) and not significantly different between women receiving HMG and rhFSH in this study. It appears that neither the amount of LH nor the content of urinary contaminants present in the HMG preparation has any significant impact on oocyte and embryo quality with regard to the morphological appearance. Our results are in agreement with the findings by Jacob et al. (1998) and Weissman et al. (1999).
A very recent meta-analysis (Agrawal et al., 2000), after re-analysing the data of randomized trials, concluded that FSH and HMG were equally effective whenever pituitary down-regulation was used. Only in cycles without pituitary down-regulation was the use of FSH associated with a higher pregnancy rate than HMG. The role of LH in folliculogenesis, oocyte function and early pregnancy outcome during down-regulated cycles has been recently challenged (Fleming et al., 1998; Filicori et al., 1999
; Westergaard et al., 2000
). The presence of LH in the gonadotrophin preparation may enhance the production of oestradiol and androgen, which play a role in oocyte maturation and embryo development.
In this study, the percentage of metaphase II oocytes, normal oocyte morphology, blastomere number and embryo grading per group were calculated. It is suggested that these parameters may be better expressed per patient since it is the patient, not the oocyte, which is the independent variable (Bergh et al., 1996). Oocyte morphology may affect the outcome of ICSI cycles (Serhal et al., 1997
) although this is not supported by others (De Sutter et al., 1996
; Balaban et al., 1998
). Zona thickness (Garside et al., 1997
) and polar body morphology (Ebner et al., 2000
) are other important parameters in assessing oocyte and embryo quality.
Morphological assessment of oocytes and embryos remains the gold standard to determine oocyte and embryo quality in clinical practice. The concentrations of hormones in the follicular fluid may reflect oocyte maturity and quality and were not examined in this study. Concentrations of oestradiol, progesterone, testosterone and LH in the follicular fluid were similar in patients who did not have features of polycystic ovaries and received HMG or rhFSH (Teissier et al., 1999). Other non-invasive tests such as early cleavage to the 2-cell stage (Shoukir et al., 1997
), development of blastocyst stage, metabolic uptake of glucose and pyruvate (Devreker et al., 2000
) may also be helpful in assessing embryo quality.
In conclusion, we found a similar percentage of metaphase II oocytes, normal morphology of zona pellucida, oocyte and polar body, blastomere number and grading of embryos after a long protocol of pituitary down-regulation and ovarian stimulation with either HMG or rhFSH for ICSI. Zona thickness and diameters of oocyte and ooplasma were also comparable in the two groups. HMG is as good as rhFSH in terms of oocyte and embryo quality.
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Notes |
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References |
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Submitted on July 12, 2000; accepted on October 30, 2000.