1 Karolinska Institutet, Department of Obstetrics and Gynaecology, Huddinge University Hospital, Stockholm, Sweden and 2 The Family Federation of Finland, Kalevagatan 16, Helsinki, Finland
3 To whom correspondence should be addressed at: Karolinska Institutet, Department of Obstetrics and Gynaecology, Huddinge University Hospital, S-141 86 Stockholm, Sweden. e-mail: Julius.Hreinsson{at}hs.se
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Abstract |
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Key words: cryopreservation/culture/maturation in vitro/oocyte/ovarian follicles
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Introduction |
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The main group of women who may benefit from the in-vitro maturation (IVM) of oocytes are those who attend for fertility treatment and who are at risk of ovarian hyperstimulation syndrome (OHSS), such as women with polycystic ovaries (PCO). As IVM is a relatively uncomplicated procedure, it may also be offered to healthy women before stimulated IVF cycles. Moreover, as well as offering rapid treatment for cryopreservation of oocytes or embryos in cancer patients, IVM is especially useful in women with the autoimmune disorder disseminated lupus erythematosus before administration of chemotherapy, as such women tolerate hormonal stimulation very poorly. IVM is also the final stage in the maturation of oocytes from ovarian primordial follicles cryopreserved within cortical tissue slices (Hovatta et al., 1996; Newton et al., 1996
; Gook et al., 1999
).
In women with PCO, hCG given before oocyte retrieval in IVM appears useful and leads to the achievement of relatively high pregnancy rates (Chian et al., 1999a; Son et al., 2002
). In these women, FSH may help in the timing of oocyte retrieval (Mikkelsen and Lindenberg, 2001a
). In the selection of optimal subjects for IVM programmes, an early follicular phase ultrasound scan has been used (Tan et al., 2002
), while the serum level of estradiol has been used as a prognostic factor (Mikkelsen et al. 2001
). The highest numbers of oocytes have been obtained from women with PCO (Child et al., 2001
).
The final stages of oocyte maturation in vivo are induced by a rise in serum LH levels. When oocytes are matured in vitro, both FSH and purified urinary gonadotrophins are added to the culture medium, and maternal serum is also used as a supplement in clinical IVM programmes (Mikkelsen et al., 1999). The signal for maturation is mediated through a common receptor for LH and chorionic gonadotrophin (CG), the LH/CG receptor, which is situated on the granulosa cells surrounding the oocyte. The binding of gonadotrophins to the receptor induces an intracellular rise in cyclic AMP levels (Conti, 2002
). Comparison of recombinant LH and hCG for oocyte maturation in clinical IVM offers the possibility of investigating any differences in the effects of these gonadotrophins in a well-defined in-vitro system, whilst simultaneously observing clinical parameters.
In order to offer optimal treatment to women at risk of OHSS, to avoid cumbersome FSH stimulation before IVF/ICSI, and to be able to carry out the full procedure from primordial follicles to live-born infants in the future, the present authors have commenced an IVM programme at their institution.
In the present study, a comparison was carried out of the natural oocyte maturation signal, LH, and hCG, which traditionally has been used to induce ovulation in assisted conception treatment, using highly purified recombinant gonadotrophins. A standard low-dose FSH priming was used to initiate follicular development, below the level of risking OHSS.
Results from parallel IVM treatments, individually optimized for women who were previously included in the study, have also been reported in order to provide a more complete information with regard to the overall success of IVM treatment in the present authors hands.
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Materials and methods |
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Randomization of the women among groups was achieved by manual blind mixing of closed envelopes using a block design with 10 treatments per block. The sequence was generated in the laboratory before information on patients attending for treatment was obtained, and concealed until intervention was assigned. For the study, subjects were randomized for oocyte maturation either in culture medium containing recombinant hCG (n = 36) or using recombinant LH (n = 37). In parallel with these treatments, 38 individually tailored IVM cycles were carried out among 32 couples, two-thirds of which were repeated cycles among couples previously included in the study. The remaining one-third of cycles were unable to adhere to the study protocol in all details regarding FSH priming.
The age of women included in the study ranged from 23 to 38 years; the mean age was 31.3, 31.9 and 31.5 years in the hCG, LH and parallel groups respectively (P = NS). Indications for treatment were in most cases unexplained infertility, anovulation or male factor infertility (Table I).
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Hormone treatment and oocyte retrieval
Women received 37.5 IU of recombinant FSH (rec-FSH) (Gonal F®; Serono Nordic, Stockholm, Sweden) on cycle days 2 to 6 after spontaneous or Gestapuran®-induced bleeding (10 mg/day for 10 days; Leo Pharma, Malmo, Sweden). In 14 cases the FSH injections were continued for an additional 2 to 11 days in order to obtain follicles suitable for aspiration. One woman received a daily dose of 75 IU rec-FSH on cycle days 2 to 8. Ultrasound monitoring was performed initially on cycle days 3 to 6 to check for cysts, and then on cycle days 6 to 8 for measurement of endometrial thickness and follicular size. Thereafter, ultrasound monitoring was performed every 23 days and oocyte retrieval was performed when at least one follicle had reached a diameter of 1014 mm and endometrial thickness was at least 5 mm. The follicles were aspirated by using a standard single-lumen needle (1.4 mm outer diameter, 1 mm inner diameter; Swemed Lab International AB, Billdal, Sweden) and low-level continuous vacuum. No flushing was carried out. Oocyte retrieval was performed with repeated punctures throughout the ovary. No hCG was administered to any of the women before oocyte retrieval.
In the individually tailored parallel group, there were 16 FSH-stimulated cycles, which were preferred for those women who did not have regular menstruations. In 12 cases the women received 37.5 IU/day rec-FSH on cycle days 2 to 8, two received 75 IU/day on cycle days 2 to 6, one woman received 50 IU/day on cycle days 2 to 6, and one woman received 200 IU/day on cycle days 2 to 6 with one additional day of 100 IU. Twenty-two were natural cycles, without FSH stimulation. No hCG was administered before oocyte retrieval during these cycles. Otherwise, clinical management was the same for the study groups and the parallel group.
Oocyte collection, maturation, fertilization and embryo culture
The follicle aspirates were collected in Falcon tubes (Becton-Dickinson, Franklin Lakes, NJ, USA) with 11.5 ml HEPES-buffered medium (Gamete-100; Vitrolife, Gothenburg, Sweden). The aspirates were filtered through a Falcon cell strainer of 70 µm mesh size (Becton-Dickinson), with the filter kept immersed in medium (Gamete-100) in order to prevent oocytes from drying out. The oocytes were washed in Gamete-100 medium and then transferred to IVF medium (IVF-100; Vitrolife) until final preparation of the maturation medium was complete.
The maturation medium consisted of Tissue Culture Medium 199 (TCM-199; Invitrogen-Gibco, Paisley, UK) supplemented (10%) with serum collected from the subject involved on the morning of oocyte retrieval, 0.3 mmol/l pyruvate, rec-FSH (0.075 IU/ml), penicillin-G (0.05 mg/ml) and streptomycin sulphate (0.075 mg/ml). For the randomized study, either recombinant hCG (0.5 IU/ml; Ovitrelle®; Serono Nordic, Stockholm, Sweden) or recombinant LH (0.5 IU/ml; Luveris®; Serono Nordic) was added to the medium. For the non-randomized parallel treatments, hCG (0.5 IU/ml; Profasi®; Serono Nordic) was added to the culture medium; otherwise, the laboratory procedures were identical for the study groups and the parallel group. Finally, the medium was filtered through a 0.2 µm sterile filter (Gelman Acrodisc, Ann Arbor, MI, USA); the filter was flushed before collecting medium for culture. Oocytes were transferred to this medium after it had been equilibrated to 37°C and pH 7.3. The time for IVM was 3236 h, and all oocytes were cultured in maturation medium, irrespective of their morphology at oocyte retrieval.
Following the maturation period, the cumulus cells were removed by incubation with hyaluronidase (80 IU/ml; Hyase; Vitrolife) for up to 1 min and denuded by pipetting. After washing, the oocytes were transferred to IVF medium (IVF-100) in 20 µl droplets under oil. Maturation in this study was defined as nuclear maturation from germinal vesicle to metaphase II (MII) stage, as identified by extrusion of the first polar body. MII oocytes were inseminated by microinjection (ICSI). Fertilized oocytes displaying two pronuclei at 1618 h after microinjection were selected for further culture until embryo transfer on day 23 after ICSI. Embryo selection for transfer or cryopreservation was performed according to previously published criteria (Fridström et al., 1999).
Embryo transfer and luteal phase support
At 1 h after oocyte retrieval the women commenced oral estradiol supplementation (Progynon®; Schering AG, Berlin, Germany), 6 mg daily. On the day after oocyte retrieval, vaginal progesterone (Progesteron MIC®; Apoteket, Sweden) was added, 400 mg in the evening and thereafter 400 mg three times daily. A maximum of two embryos was transferred at a time, using a soft catheter and ultrasonographic guidance.
Estradiol and progesterone administration were continued until pregnancy testing. If the result was positive, the hormonal substitution was continued until the ninth gestational week. Pregnancy testing was performed using a urinary assay (SureStick®; Applied Biotech, Inc., CA, USA) at 1416 days after embryo transfer. Negative results were confirmed by registering menstrual bleeding, and positive results by abdominal ultrasound examination at 4 and 6 weeks after oocyte retrieval. Only clinical pregnancies confirmed by ultrasonography were recorded.
Power calculation and statistical analysis
According to power calculations, a minimum of 160 oocytes in each arm of the study would have been needed to show a significant difference in oocyte maturation rate, which was the primary outcome measure of this randomized study, from 60 to 75%. These levels were based on previous publications on the subject. The significance level (alpha) was set at 0.05 with a power of 80%. It was estimated that about 80 women would be needed for the study, and the required number of oocytes was reached when 73 women had been randomized. At that stage, an interim analysis of the number of oocytes obtained showed that sufficient numbers were already available to reach the required power level. Hence, recruitment was stopped and an analysis of the results commenced.
The chi-square test and Fishers exact test were used to test differences between groups with regard to different rates of development. A P-value < 0.05 was considered statistically significant.
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Results |
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In the recombinant hCG group, 29% of patients who underwent embryo transfer had at least one good quality embryo for transfer, and 46% had at least one 4-cell embryo on day 2 or one 8-cell embryo on day 3 after ICSI. For the LH group, these values were 24 and 28% respectively (P = NS). In the parallel group, the proportion of patients with at least one good quality embryo was 48%, and 52% had at least one 4-cell embryo on day 2 or at least one 8-cell embryo on day 3 after ICSI. These values were lower than observed in the authors regular IVF/ICSI programme, where most patients produce at least one embryo of good quality and good cleavage progression for embryo transfer (results not shown).
Failed maturation was observed in four cases in the study group; two of these patients had one oocyte, one patient had two oocytes, and one had eight oocytes. Two patients in the parallel group had failed to achieve oocyte maturation, including one patient with one oocyte and one with three oocytes.
Cryopreservation of embryos was performed in one case in the study group and in three cases among the subjects not included in the study. To date, no pregnancies have been achieved after thawing of these embryos, though one couple still has frozen embryos because of a pregnancy from the original cycle.
The four pregnancies resulting from the randomized hCG and LH treatments proceeded uneventfully, whereas two of the seven pregnancies in the parallel group ended in miscarriage (one in the first trimester, and one in the second trimester). One pregnancy in the randomized treatment groups showed two implantation sacs, one of which was viable; all other pregnancies were singleton. Subsequently, nine healthy infants have been born (six girls and three boys).
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Discussion |
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Whilst hCG has been administered by intramuscular injection to subjects undergoing IVM in cases of polycystic ovary syndrome (PCOS) (Chian et al., 1999b; Child et al., 2001
), FSH was not administered in these studies. Follicle priming with FSH without hCG injection has also been shown to be effective in women with PCOS (Mikkelsen and Lindenberg, 2001a
). In the present study, only a small proportion of women with confirmed PCOS were enrolled, although 12 of those in the recombinant LH group had anovulation as an indication for treatment. An analysis of subgroups of patients with different indications for treatment did not seem meaningful in the present study, however.
The technique of IVM is different from that of standard IVF, both in terms of clinical management and laboratory techniques. The first pregnancy was achieved 6 months after the study was initiated, although meiotically competent oocytes were obtained over the whole study period. Higher success rates were also achieved with individually tailored treatments for women previously included in the randomized study. This may in part be explained by previous experience of monitoring the subjects and, on that basis, the following treatments were either natural or FSH-primed cycles. The need for FSH stimulation in IVM cycles has been shown to be important for women with PCOS (Mikkelsen and Lindenberg, 2001), and higher total doses of FSH were used for some of the parallel treatments.
Low-dose FSH priming has been used with good results in terms of oocyte maturation and cleavage rates (Suikkari et al., 2000); hence, the decision was made to continue with this regimen. The maturation time of 3236 h is practically applicable in a clinical setting, and maturation times of 2836 h have been shown previously to be adequate in IVM (Mikkelsen et al., 2001
). Oocytes have also been shown previously to be responsive to recombinant gonadotrophins in vitro (Anderiesz et al., 2000
), using 1:10 ratios of FSH:LH. The levels used in the present study were chosen on the basis of previous experience in IVM (Suikkari et al., 2000
).
As patient serum is a necessary component of the maturation medium for IVM, it was collected and processed on the day of oocyte retrieval. Although levels of FSH, LH or estradiol were not monitored in serum used for the maturation medium, oocyte retrieval was (on average) performed on cycle day 10, well before the natural LH surge, and no premature luteinization of the oocytes or cumulus cells was observed. In addition, the groups were treated in the same way, apart from the addition of recombinant hormones to the maturation medium.
The maturation medium used in the study, TCM-199, is rich and has a composition which is different from that of the Vitrolife culture medium used for oocyte and embryo culture after ICSI. It is unclear whether this might affect the oocytes in any way, although sequential media with different compositions are used regularly in IVF where the different stages of development each have varied requirements. More importantly, the present authors have extensive experience in use of the Vitrolife media range for regular IVF/ICSI treatments.
Ten of the women included in the present study had a previous history of a poor response after conventional IVF/ICSI, or previous failed IVF/ICSI cycles, and this may in part explain the modest pregnancy rates observed. Selection criteria that might have reflected diminished ovarian reserve, such as elevated FSH levels or decreased inhibin B levels in serum (Mikkelsen et al., 2000), were not used. More efficient IVM treatments may possibly be achieved by using a stricter selection of subjects. However, previous failed IVF/ICSI cycles did not preclude pregnancy for individual women in the present study.
In some cases, a poorer embryo quality was observed compared with the regular IVF/ICSI programme, as well as a slower cleavage of the embryos. A possible explanation for this might be asynchrony between nuclear and cytoplasmic maturation (Combelles et al., 2002). The importance of cytoplasmic maturation and acquisition of cytoplasmic proteins necessary for development of the embryo has been recognized (Trounson et al., 2001
), and the dialogue between the oocyte and granulosa/cumulus cells is a critical determinant for correct expression of the genes coding for these proteins (Eichenlaub-Ritter and Peschke, 2002
).
The timing of oocyte aspiration may be critical, allowing as many follicles as possible to reach sufficient size for cytoplasmic competence of the oocytes (Trounson et al., 2001) while avoiding a prolonged negative effect of the developing dominant follicle. Suppression by the dominant follicle seems to be an endocrine rather than a paracrine effect, as retrieval and IVM of oocytes is comparable from ipsilateral and contralateral ovaries with regard to the dominant follicle (Mikkelsen and Lindenberg, 2001b
).
The need for optimization of different varieties of IVM for different groups of subjects is apparent. In the present study the best pregnancy rates were achieved when the treatments were adjusted to individuals and not carried out according to a rigid scheme. Natural cycles without FSH priming in regularly menstruating women have been shown previously to work equally well as FSH-primed cycles (Mikkelsen et al., 1999), whereas different regimens may be needed for anovulatory women, particularly those with PCO.
In conclusion, no significant differences were observed when using either recombinant hCG or LH for oocyte maturation in IVM treatments, with regard to both embryological and clinical parameters. The present results highlight the importance of optimizing IVM for different groups of subjects, as after individualized IVM treatment a relatively good pregnancy rate per embryo transfer of 23% can be achieved.
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Acknowledgements |
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References |
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Submitted on February 6, 2003; resubmitted on May 6, 2003; accepted on July 7, 2003.