Recombinant LH is equally effective as recombinant hCG in promoting oocyte maturation in a clinical in-vitro maturation programme: a randomized study

Julius Hreinsson1,3, Björn Rosenlund1, Barbro Fridén1, Lev Levkov1, Ingvar Ek1, Anne-Maria Suikkari2, Outi Hovatta1 and Margareta Fridström1

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Fertilization treatment using oocytes matured in vitro from pre-ovulatory follicles has many potential applications. It minimizes the risk of severe ovarian hyperstimulation and is an alternative for women with polycystic ovary syndrome who may have problems regarding stimulation for IVF. In-vitro maturation (IVM) may prove important for subjects needing fertility preservation, and also provides information about the final stages of oocyte maturation. METHODS: From a randomized study of 73 women in an IVF programme, 36 subjects with 228 oocytes were allocated for oocyte maturation in culture medium with recombinant hCG, and 37 subjects with 256 oocytes for maturation with recombinant LH. The primary outcome was the rate of nuclear maturation of oocytes to metaphase II. During the same period, 32 women outside the study underwent 38 individually tailored IVM treatments. RESULTS: The oocyte maturation rate was 54.8% with hCG and 55.9% with LH; fertilization and cleavage rates were not significantly different. Three pregnancies were achieved in the hCG group and one in the LH group. Seven pregnancies (22.6% per embryo transfer) were achieved in the parallel group. CONCLUSIONS: Recombinant hCG or LH are equally effective in promoting oocyte maturation in a clinical IVM programme.

Key words: cryopreservation/culture/maturation in vitro/oocyte/ovarian follicles


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Oocytes of several animal species undergo spontaneous maturation when they are removed from their follicles (Edwards, 1965Go). The first children (triplets) originating from in-vitro-matured oocytes after extraction from ovarian tissue were born in 1991 (Cha et al., 1991Go), since when this technique has been further developed and many more healthy children have been born from in-vitro-matured oocytes (Barnes et al., 1995Go; Cha and Chian, 1998Go; Chian et al., 1999aGo; b, 2001; Jaroudi et al., 1999Go; Mikkelsen et al., 1999Go; Mikkelsen and Lindenberg, 2001aGo; b; Suikkari et al., 2000Go; Child et al., 2001Go; Tan et al., 2002Go). The cryopreservation of embryos using oocytes matured in vitro has also resulted in normal pregnancies (Suikkari et al., 2000Go; Chian et al., 2001Go).

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., 1996Go; Newton et al., 1996Go; Gook et al., 1999Go).

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., 1999aGo; Son et al., 2002Go). In these women, FSH may help in the timing of oocyte retrieval (Mikkelsen and Lindenberg, 2001aGo). In the selection of optimal subjects for IVM programmes, an early follicular phase ultrasound scan has been used (Tan et al., 2002Go), while the serum level of estradiol has been used as a prognostic factor (Mikkelsen et al. 2001Go). The highest numbers of oocytes have been obtained from women with PCO (Child et al., 2001Go).

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., 1999Go). 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, 2002Go). 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Subjects
Patients were recruited from an IVF programme, with inclusion criteria being the same as for regular IVF or ICSI treatments; that is, healthy women aged between 20 and 40 years, with an indication for fertility treatment requiring assisted reproduction. Couples with male factor infertility requiring sperm extraction from a testicular biopsy were not considered for the study. A total of 84 couples was assessed for eligibility for the randomized study, all of whom previously provided their informed consent to participate and receive treatment. Eleven cycles were cancelled before oocyte retrieval (OR); in most cases this was due to suboptimal endometrial or follicular development. In total, 73 women completed the treatment course.

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).


View this table:
[in this window]
[in a new window]
 
Table I. Age and indication for treatment among the women randomized for participation in the study or coming for parallel individually tailored treatments
 
The study was approved by the Ethics Committee of the Karolinska Institutet at Huddinge University Hospital.

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 2–3 days and oocyte retrieval was performed when at least one follicle had reached a diameter of 10–14 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 1–1.5 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 32–36 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 16–18 h after microinjection were selected for further culture until embryo transfer on day 2–3 after ICSI. Embryo selection for transfer or cryopreservation was performed according to previously published criteria (Fridström et al., 1999Go).

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 14–16 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 Fisher’s 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.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A total of 228 oocytes was collected from women randomized for oocyte maturation in culture medium with recombinant hCG, and 256 oocytes from women randomized for maturation with recombinant LH. Details regarding the cycle day for oocyte retrieval, numbers of oocytes per retrieval and progression to embryo transfer are listed in Table II. The mean number of oocytes obtained per oocyte retrieval was 6.3 in the hCG group, and 6.9 in the LH group.


View this table:
[in this window]
[in a new window]
 
Table II. Details of cycle day for oocyte retrieval (OR), numbers of oocytes per retrieval and progression to embryo transfer (ET) among women randomized for oocyte maturation with either recombinant hCG or LH, or attending for IVM treatment parallel to the study
 
When cultured with recombinant hCG, 55% of the oocytes matured to MII, while 56% of those cultured with LH reached MII (P = NS). No significant differences were observed in terms of oocyte maturation rate, fertilization rate or embryo cleavage between the groups. The detailed results of oocyte maturation and embryo culture are listed in Table III. Most of the subjects attending for oocyte retrieval reached embryo transfer (78% with hCG, 68% with LH; P = NS). Three clinical pregnancies were achieved in the hCG-maturation group and one in the LH group (P = NS).


View this table:
[in this window]
[in a new window]
 
Table III. Results of oocyte retrieval, maturation and embryo culture among women randomized for oocyte maturation with recombinant hCG or LH, or attending for in-vitro maturation (IVM) treatment parallel to the study
 
In the parallel treatment group (which was not included in the randomized study), a total of 226 oocytes was retrieved from 38 oocyte retrievals, while 59% of the oocytes matured and 82% of the cycles progressed to embryo transfer. The pregnancy rate in the parallel group was higher than in either study group (23% per transfer). Complete results are listed in Tables II and III.

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).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It has been shown in the present study that recombinant hCG is as effective as recombinant LH in promoting oocyte maturation in clinical IVM treatment. This was not an unexpected finding, as the same receptor on the granulosa cells is responsible for the effect of both LH and hCG on oocyte maturation, which may include reduced transfer of maturation-arresting substances such as cAMP to the oocyte (Eppig, 1991Go). However, this defined in-vitro system provides a unique opportunity to study any functional differences between these two recombinant gonadotrophins in a clinical setting. Since the study was powered for oocyte maturation, results regarding fertilization, cleavage and pregnancy rates must be interpreted with caution. Although the present results do not show any such differences, the finding is interesting since both LH and hCG have been used previously for oocyte maturation in vitro (Mikkelsen et al., 1999Go; Suikkari et al., 2000Go). Herein, comparable results were obtained with regard to oocyte maturation as were found in earlier studies.

Whilst hCG has been administered by intramuscular injection to subjects undergoing IVM in cases of polycystic ovary syndrome (PCOS) (Chian et al., 1999bGo; Child et al., 2001Go), 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, 2001aGo). 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, 2001Go), 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., 2000Go); hence, the decision was made to continue with this regimen. The maturation time of 32–36 h is practically applicable in a clinical setting, and maturation times of 28–36 h have been shown previously to be adequate in IVM (Mikkelsen et al., 2001Go). Oocytes have also been shown previously to be responsive to recombinant gonadotrophins in vitro (Anderiesz et al., 2000Go), 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., 2000Go).

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., 2000Go), 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., 2002Go). The importance of cytoplasmic maturation and acquisition of cytoplasmic proteins necessary for development of the embryo has been recognized (Trounson et al., 2001Go), 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, 2002Go).

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., 2001Go) 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, 2001bGo).

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., 1999Go), 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.


    Acknowledgements
 
Recombinant LH and hCG were kindly donated by Serono Nordic, Stockholm, Sweden. The authors thank the midwives and technicians at the Fertility Unit for their help in the study, and Dr Nicholas Bolton for language revision of the manuscript.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Anderiesz, C., Ferraretti, A., Magli, C., Fiorentino, A., Fortini, D., Gianaroli, L., Jones, G.M. and Trounson, A.O. (2000) Effect of recombinant human gonadotrophins on human, bovine and murine oocyte meiosis, fertilization and embryonic development in vitro. Hum. Reprod., 15, 1140–1148.[Abstract/Free Full Text]

Barnes, F.L., Crombie, A., Gardner, D.K., Kausche, A., Lacham-Kaplan, O., Suikkari, A.M., Tiglias, J., Wood, C. and Trounson, A.O. (1995) Blastocyst development and birth after in vitro maturation of human primary oocytes, intracytoplasmic sperm injection and assisted hatching. Hum. Reprod., 10, 3243–3247.[Abstract]

Cha, K.Y. and Chian, R.C. (1998) Maturation in vitro of immature human oocytes for clinical use. Hum. Reprod. Update, 4, 103–120.[Abstract/Free Full Text]

Cha, K.Y., Koo, J.J., Ko, J.J., Choi, D.H., Han, S.Y. and Yoon, T.K. (1991) Pregnancy after in vitro fertilization of human follicular oocytes collected from nonstimulated cycles, their culture in vitro and their transfer in a donor oocyte program. Fertil. Steril., 55, 109–113.[ISI][Medline]

Chian, R.C., Buckett, W.M., Too, L.L. and Tan, S.L. (1999a) Pregnancies resulting from in vitro matured oocytes retrieved from patients with polycystic ovary syndrome after priming with human chorionic gonadotropin. Fertil. Steril., 72, 639–642.[CrossRef][ISI][Medline]

Chian, R.C., Gulekli, B., Buckett, W.M. and Tan, S.L. (1999b) Priming with human chorionic gonadotropin before retrieval of immature oocytes in women with infertility due to the polycystic ovary syndrome. N. Engl. J. Med., 341, 1624–1626.[Free Full Text]

Chian, R.C., Gulekli, B., Buckett, W.M. and Tan, S.L. (2001) Pregnancy and delivery after cryopreservation of zygotes produced by in-vitro matured oocytes retrieved from a woman with polycystic ovarian syndrome. Hum. Reprod., 16, 1700–1702.[Abstract/Free Full Text]

Child, T.J., Abdul-Jalil, A.K., Gulekli, B. and Tan, S.L. (2001) In vitro maturation and fertilization of oocytes from unstimulated normal ovaries, polycystic ovaries, and women with polycystic ovary syndrome. Fertil. Steril., 76, 936–942.[CrossRef][ISI][Medline]

Combelles, C.M., Cekleniak, N.A., Racowsky, C. and Albertini, D.F. (2002) Assessment of nuclear and cytoplasmic maturation in in-vitro matured human oocytes. Hum. Reprod., 17, 1006–1016.[Abstract/Free Full Text]

Conti, M. (2002) Specificity of the cyclic adenosine 3',5'-monophosphate signal in granulosa cell function. Biol. Reprod., 67, 1653–1661.[Abstract/Free Full Text]

Edwards, R.G. (1965) Maturation in vitro of mouse, sheep, cow, pig, rhesus monkey and human ovarian oocytes. Nature, 208, 349–351.[ISI][Medline]

Eichenlaub-Ritter, U. and Peschke, M. (2002) Expression in in-vivo and in-vitro growing and maturing oocytes: focus on regulation of expression at the translational level. Hum. Reprod. Update, 8, 21–41.[Abstract/Free Full Text]

Eppig, J.J. (1991) Intercommunication between mammalian oocytes and companion somatic cells. BioEssays, 13, 569–574.[ISI][Medline]

Fridström, M., Carlström, K., Sjöblom, P. and Hillensjö, T. (1999) Effect of prednisolone on serum and follicular fluid androgen concentrations in women with polycystic ovary syndrome undergoing in-vitro fertilization. Hum. Reprod., 14, 1440–1444.[Abstract/Free Full Text]

Gook, D.A., Edgar, D.H. and Stern, C. (1999) Effect of cooling rate and dehydration regimen on the histological appearance of human ovarian cortex following cryopreservation in 1,2-propanediol. Hum. Reprod., 14, 2061–2068.[Abstract/Free Full Text]

Hovatta, O., Silye, R., Krausz, T., Abir, R., Margara, R., Trew, G., Lass, A. and Winston, R.M.L. (1996) Cryopreservation of human ovarian tissue using dimethylsulphoxide and propanediol-sucrose as cryoprotectants. Hum. Reprod., 11, 1268–1272.[Abstract]

Jaroudi, K.A., Hollanders, J.M., Elnour, A.M., Roca, G.L., Atared, A.M. and Coskun, S. (1999) Embryo development and pregnancies from in-vitro matured and fertilised human oocytes. Hum. Reprod., 14, 1749–1751.[Abstract/Free Full Text]

Mikkelsen, A.L. and Lindenberg, S. (2001a) Benefit of FSH priming of women with PCOS to the in vitro maturation procedure and the outcome: a randomized prospective study. Reproduction, 122, 587–592.[Abstract/Free Full Text]

Mikkelsen, A.L. and Lindenberg, S. (2001b) Influence of the dominant follicle on in-vitro maturation of human oocytes: a prospective non-randomized study. R. B. M. Online, 3, 199–204.

Mikkelsen, A.L., Smith, S.D. and Lindenberg, S. (1999) In-vitro maturation of human oocytes from regularly menstruating women may be successful without follicle stimulating hormone priming. Hum. Reprod., 14, 1847–1851.[Abstract/Free Full Text]

Mikkelsen, A.L., Smith, S. and Lindenberg, S. (2000) Impact of oestradiol and inhibin A concentrations on pregnancy rate in in-vitro oocyte maturation. Hum. Reprod., 15, 1685–1690.[Abstract/Free Full Text]

Mikkelsen, A.L., Andersson, A.M., Skakkebaek, N.E. and Lindenberg, S. (2001) Basal concentrations of oestradiol may predict the outcome of in-vitro maturation in regularly menstruating women. Hum. Reprod., 16, 862–867.[Abstract/Free Full Text]

Newton, H., Aubard, Y., Rutherford, A., Sharma, V. and Gosden, R. (1996) Low temperature storage and grafting of human ovarian tissue. Hum. Reprod., 11, 1487–1491.[Abstract/Free Full Text]

Son, W.Y., Yoon, S.H., Lee, S.W., Ko, Y., Yoon, H.G. and Lim, J.H. (2002) Blastocyst development and pregnancies after IVF of mature oocytes retrieved from unstimulated patients with PCOS after in-vivo HCG priming: case report. Hum. Reprod., 17, 134–136.[Abstract/Free Full Text]

Suikkari, A.M., Tulppala, M., Tuuri, T., Hovatta, O. and Barnes, F. (2000) Luteal phase start of low-dose FSH priming of follicles results in efficient recovery, maturation and fertilization of immature human oocytes. Hum. Reprod., 15, 747–751.[Abstract/Free Full Text]

Tan, S.L., Child, T.J. and Gulekli, B. (2002) In vitro maturation and fertilization of oocytes from unstimulated ovaries: predicting the number of immature oocytes retrieved by early follicular phase ultrasonography. Am. J. Obstet. Gynecol., 186, 684–689.[CrossRef][ISI][Medline]

Trounson, A., Anderiesz, C. and Jones, G. (2001) Maturation of human oocytes in vitro and their developmental competence. Reproduction, 121, 51–75.[Abstract/Free Full Text]

Submitted on February 6, 2003; resubmitted on May 6, 2003; accepted on July 7, 2003.