Department of Gynecology and Obstetrics, Medical University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany
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Abstract |
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Key words: GnRH analogue/ICSI/IVFembryo transfer/oocyte quality/polycystic ovarian syndrome
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Introduction |
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Several retrospective (Dor et al., 1992; Urman et al., 1992
; Balen et al., 1993
; Homburg et al., 1993a
,b
; Kodama et al., 1995
) and prospective controlled studies (Salat-Baroux et al., 1988
; Ashkenazi et al., 1989
) have tried to answer the question: do PCOS patients have the same outcome in in-vitro fertilization (IVF) treatment cycles, compared to non-PCOS patients suffering only from tubal infertility? The controversial question of whether there is a benefit of pituitary suppression for PCOS patients in an IVF programme is discussed in this study.
It has been claimed that oocyte maturation is disturbed in PCOS patients. However, there are no data at the time of writing on oocyte maturity, i.e. the absolute number and rate of metaphase II oocytes in these patients, since in IVF cycles only cumulus oocyte complexes can be morphologically analysed. The data on embryo quality in PCOS patients in relation to pregnancy and abortion rates are poor.
In this study we retrospectively compared the IVF data in PCOS patients to an age-matched control group. The main indication for IVF was a male factor and therefore, in all cases, intracytoplasmic sperm injection (ICSI) was performed. This gave us the unique opportunity to validate the rate of metaphase II oocytes after removal of the cumulus and corona cells. Laboratory and clinical data from these patients were compared.
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Materials and methods |
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Control group
For the control group two patients per PCOS patient were matched. We looked for the first patient being of approximately the same age (±3 months) forward (patient 1) and backward (patient 2) in time. The clinical characteristics, which were noted from the files, are shown in Table I.
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Ovulation induction was done by administration of 10 000 IU of human chorionic gonadotrophin (HCG) at an average leading follicle diameter of 2022 mm and an adequate concentration of oestradiol. Oocyte retrieval was carried out under general anaesthesia if required, using vaginal ultrasound-guided puncture of follicles 36 h after HCG application.
Gamete preparation and ICSI procedure
Sperm preparation was carried out using the `mini swim-up' method in cases of severe male factor infertility. We use this technique of sperm preparation in preference to Percoll because it has been proven to be effective for the isolation of spermatozoa even in the presence of severely impaired semen parameters (Al-Hasani et al., 1995). In each semen sample used, motile spermatozoa were present. Oocytes were treated with 0.5% hyaluronidase (Sigma Co., Hamburg, Germany) for 1015 s for enzymatic lysis of the cumulus oophorus cells. Cells of the corona radiata were removed mechanically with a Pasteur pipette under stereomicroscopic guidance (magnification x50). Subsequently, the maturity of the oocytes was determined. ICSI was performed as previously described (Al-Hasani et al., 1995
).
Assessment of embryo quality
Embryos were scored in accordance to a modified form of the previously published cumulative embryo score (Steer et al., 1992). Briefly, the number of blastomeres of each embryo was multiplied with the grade of the morphological appearance of the embryo. The gradings were 3 (ideal quality: i.e. regular blastomeres, no fragments), 2 (moderate: i.e. irregular blastomeres, no fragments), and 1 (irregular: fragments present). All individual scores of the transferred embryos were summarized and resulted in the modified cumulative embryo score.
Statistics
Mean values were compared using the MannWhitney test. Percentages were compared using Pearson's 2 test. A two-tailed asymptotic significance of <0.05 was interpreted as being statistically significant. Ratios, e.g. the ratio of metaphase II oocytes to the total number of oocytes, were compared using the Sokal test (Sokal and Rohlf, 1969
).
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Results |
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Regarding the course of the stimulation, there was a significant lower mean number of ampoules needed in the PCOS patients, compared to the controls (34.88 versus 42.37, P < 0.01). Despite that, mean oestradiol values on the day of HCG were significantly higher (3843.59 versus 2367.40 pg/ml, P < 0.01) and the mean oocyte number significantly higher in the PCOS group compared to the controls (15.18 versus 10.07, P < 0.01). The rate of metaphase II oocytes (53.48% versus 62.66%), the rate of germinal vesicle oocytes (4.63% versus 7.45%) and the fertilization rate of metaphase II oocytes (62.66% versus 56.42%) were not different between PCOS and non-PCOS patients (Table II).
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Discussion |
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The incidence of PCOS patients in this study (3%) is extremely low, compared to the incidence published in other studies. Polson et al. (1988), Clayton et al. (1992) and Farquhar et al. (1994) found an incidence of 23, 22 and 21% respectively for PCOS patients according to the Adams criteria (Adams et al., 1985). However Jacobs (1992) made the criticism that these collectives did not represent the general population, because only
20% of those who were initially selected to be recruited were finally included in the analysis. He thought it was likely that mainly women suffering from endocrinological problems would be interested in being included in such studies. Our patients were mainly those who suffered from severe male factor infertility, therefore it may be that most PCOS patients were treated by other techniques, such as ovulation induction or insemination.
One of the main characteristics of PCOS patients is the high tonic LH concentration, which exceeds that of the serum FSH concentration. This high LH concentration is thought to be detrimental to oocyte maturation and quality and can be the cause of the lower conception rate and higher abortion rate in these patients (Adams et al., 1985; Howles et al., 1986
; Homburg et al., 1988
; Regan et al., 1990
). A similar observation was made in IVF patients with a premature LH surge (Punnonen et al., 1988
). Therefore the authors concluded that pituitary suppression could be helpful in these patients.
Until now, no data have existed on the rate of metaphase II oocytes in PCOS patients undergoing an IVF or IVF/ICSI treatment. Many authors claimed to have studied the quality of retrieved oocytes in PCOS patients (Tanbo et al., 1990; Kodama et al., 1995
; Tarlatzis et al., 1995
). Some found a worse oocyte quality to be the cause of a low fertilization and a high abortion rate (Kodama et al., 1995
; Tarlatzis et al., 1995
).
However, all studies analysed cumulus oocyte complexes. These complexes can be graded morphologically, but show only a weak correlation with the real quality of oocytes in terms of maturity. The real rate of metaphase II oocytes can only be assessed after the removal of cumulus cells, which is not done in conventional IVF treatment. Only in ICSI cycles can the rate of metaphase II oocytes be seen before fertilization is achieved, because all cumulus cells must be removed using hyaluronidase treatment.
In a recent study Aboulghar et al. (1997) presented data on oocyte quality from IVF and ICSI cycles in PCOS and non-PCOS patients. However, this study had some other main design characteristics, compared to our own study. Principally, both studies were retrospective case control studies. The definition of PCOS in the study of Aboulghar et al. (1997), however, was done according only to sonographic criteria. The study population was not homogeneously treated by ICSI, therefore data on oocyte maturity were given only for a small number of patients. No data were given on the percentage of patients treated in the PCOS and non-PCOS groups by conventional IVF and ICSI. The authors concluded that they could show a lower number of `high quality oocytes' in PCOS compared to non-PCOS patients, and that this factor could be attributed to a lower fertilization rate in these cycles. These data differ from our own results, but may be the result of the factors outlined above, and especially the insufficient method of oocyte grading in IVF cycles.
Our data clearly show that morphological criteria do not account for the higher abortion rate, since neither the absolute number of metaphase II oocytes nor the metaphase II oocyte/total oocyte ratio was significantly different between both groups. The embryo quality and the mean number of embryos replaced was in fact higher compared to the control group.
There was no difference in the oocyte retrieval/transfer ratio in our study. This may be due to the application of the ICSI procedure in most of the cycles, which may overcome the fertilization failure in most cases and may explain the differences in the data of Kodama et al. (1995). In fact, the fertilization failure rate, defined as the ratio of cycles without any fertilization, was slightly higher in non-PCOS patients. However, the difference did not reach statistical significance.
The difference of our data to those of other authors may also be due to the high incidence of pure male factor infertility. Due to this, the rate of patients with previous fertilization failure or bad response may be lower compared to a cohort of patients treated by conventional IVF.
The first prospective, closed, randomized study to evaluate the advantage of a pituitary suppression for ovulation induction in PCOS patients was performed by Johnson and Pearce (1990). They noted the advantage of pituitary suppression regarding the abortion rate in PCOS patients. These results were confirmed by others (Balen et al., 1993; Homburg et al., 1993a
, b
).
Only two prospective trials were published on this topic. Ashkenazi et al. (1989) published data of a prospective, randomized study on a very small number of non-defined PCOS patients. They treated six patients using urinary FSH, six patients using urinary FSH and HMG and six patients using urinary FSH and HMG after pituitary suppression. They did not find any difference, not at least according to the low number of patients included.
Sixteen PCOS patients were stimulated for IVF in 17 cycles in another prospective, randomized study (Salat-Baroux et al., 1988). The control group consisted of six patients. Only 11 cycles in the PCOS group were suppressed, all other cycles were done without pituitary suppression. There was no difference in the oocyte recovery rate, fertilization or cleavage rate, number of embryos transferred or pregnancy rate. Only one severe OHSS occurred in the PCOS group and abortions were not reported.
To conclude, only two, very small prospective studies were done on PCOS patients to show differences compared to endocrinologically normal controls (Salat-Baroux et al., 1988; Ashkenazi et al., 1989
). The stimulation procedure was not homogeneous and only rarely were patients treated according to the long protocol in these two prospective studies. The larger retrospective analyses uniformly show the advantage of pituitary suppression for the treatment outcome of PCOS patients.
The data presented here show no advantage of LH suppression in PCOS patients, since despite the use of GnRH analogue treatment the incidence of clinical abortions was significantly higher in women with PCOS compared with controls. The pregnancy rate was in the same range. These data must be interpreted with respect to the higher mean number of embryos replaced per transfer in the PCOS group and the higher cumulative embryo score of these embryos. Since a higher number and better quality of embryos are positively correlated with the ongoing pregnancy rate, it is surprising that even under these circumstances, the abortion rate was significantly higher in the PCOS group.
The higher number of embryos which could be replaced was mainly due to the higher number of oocytes retrieved and a higher absolute number of 2PN oocytes in the PCOS patient group. The higher number of retrieved oocytes was also the reason for a lower fertilization failure rate. However, the last did not meet statistical significance.
One of the factors that can significantly influence the abortion rate in PCOS patients is a higher body mass index (BMI, kg/m2) (Maresh et al., 1986; Bohrer and Kemmann, 1987
; Eshel et al., 1988
; Homburg and Jacobs, 1989
; Hamilton-Fairley et al., 1992
; Franks and Hamilton-Fairley, 1994
), even if this opinion is not shared by all authors (Filicori et al., 1990
, 1991
). Since the BMI was not significantly different in the PCOS and non-PCOS group, the higher abortion rate in the PCOS group cannot be attributed to this factor.
It is a well known fact that in PCOS patients the rate of severe OHSS is higher compared with normal women. This was corroborated by the observations of our study. It may be due to higher oestradiol concentrations on the day of HCG and a higher number of follicles present on that day, expressed as a higher absolute number of oocytes retrieved.
In summary, we have found a higher rate of severe OHSS, but no difference in the rate of metaphase II oocytes and fertilization rate in patients with PCOS. The latter finding has not been reported previously. Despite a higher mean number of embryos transferred and a higher cumulative embryo score, there was a similar pregnancy and multiple pregnancy rate, and a significantly higher clinical abortion rate in PCOS patients. This demonstrates that despite a given nuclear maturity of oocytes, cytoplasmic factors might negatively influence the developmental potential of embryos and the outcome of pregnancies in PCOS patients.
The hypothesis of a cytoplasmic factor is supported by data from Sengoku et al. (1997). They performed a cytogenetic analysis on oocytes from PCOS and non-PCOS patients and could not find a higher incidence of chromosome number abnormalities in unfertilized oocytes from PCOS patients, retrieved after hormonal stimulation according to a long protocol. Interestingly, the only differences between the two groups were a higher number of retrieved oocytes and a lower fertilization rate in the PCOS group. No difference was found regarding the clinical pregnancy and miscarriage rate.
The impaired cytoplasmic oocyte maturity may be caused by a long-term exposure of oocytes to high tonic LH concentrations. Therefore it can be postulated that a longer pituitary suppression could be helpful in these patients. The data from Damario et al. (1997), who introduced a modified long protocol with oral contraceptive pills for 25 days prior to pituitary suppression using a GnRH agonist in IVF cycles, support this hypothesis. The authors found a benefit from this dual `long-term' suppression on the ongoing pregnancy rate in a retrospective analysis.
More studies, preferentially prospective and randomized, which also analyse oocyte maturity and embryo quality are needed to find the optimal protocol for this group of patients.
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Acknowledgments |
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Notes |
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References |
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Submitted on July 1, 1998; accepted on October 16, 1998.