Basal concentrations of oestradiol may predict the outcome of in-vitro maturation in regularly menstruating women

A.L. Mikkelsen1,3, A.-M. Andersson2, N.E. Skakkebæk2 and S. Lindenberg1

1 The Fertility Clinic, Institute of Human Reproduction, 3 Fruebjergvej, DK-2100 Copenhagen and 2 Department of Growth and Reproduction, Copenhagen University Hospital, 9 Blegdamsvej, DK-2100 Copenhagen, Denmark


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Retrospectively it was examined whether the number of retrieved oocytes, the maturation rate and cleavage rate can be predicted in regularly menstruating women by the use of the following predictive variables on cycle day 3–4: the concentration of FSH, oestradiol, inhibin B and inhibin A in serum and and the number of ovarian follicles seen by vaginal ultrasound. The study included 132 consecutive aspirations in 100 women attending the clinic for in-vitro maturation due to male factor and/or tubal factor. Fifteen pregnancies were obtained after transfer in 83 cycles, giving a pregnancy rate of 15/132 (11%) per aspiration and 15/83 (18%) per transfer. The concentration of FSH and the number of follicles on day 3 predicted the number of oocytes retrieved, whereas these parameters did not predict the subsequent development of oocytes. No correlation was found between the inhibin B, inhibin A, oestradiol and the number of oocytes respectively. The group with a low concentration of oestradiol on cycle day 3 (threshold <200 pmol/l) (group 1, n = 106 cycles) had a significantly higher pregnancy rate compared to the group with a higher concentration (group 2, n = 26 cycles) (14 versus 0% per aspiration, P = 0.03). The group with a low concentration of oestradiol was subdivided according to the concentration of inhibin A. Group 1a: low inhibin A (threshold <10 pg/ml, n = 84 cycles) and group 1b: high inhibin A concentration (>=10 pg/ml, n = 19). The pregnancy rate in group 1a (14/84, 17%) differed significantly from group 1b (0/19, 0%) (P = 0.03). It is concluded that a low basal concentration of oestradiol (<200 pmol/l) was shown to be a useful prognostic factor of pregnancy in IVM. The concentration of inhibin A (<10 pg/ml) was of added value.

Key words: human/maturation in vitro/oocytes/predictive variables


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Basal hormone concentrations on day 3 of a natural cycle have previously been accepted as good markers for reproductive performance. High basal FSH concentrations (>15 IU/l) (Scott et al., 1989Go) and low inhibin B concentrations (<45 pg/ml) (Seifer et al., 1997Go) have been shown to be associated with low ovarian reserve.

The basal FSH concentration has proved to be a reliable prognostic factor in ovarian stimulation and IVF (Muasher et al., 1988Go; Scott et al., 1989Go; Evers et al., 1998Go). In addition, elevated concentrations of oestradiol predicted poor response (Licciardi et al., 1995Go; Evers et al., 1998Go). Transvaginal ultrasound determination of either the number of follicles of 2–5 mm diameter or the ovarian volume has been reported to predict the number of oocytes retrieved and the pregnancy rate in conventional IVF (Syrop et al., 1995Go, 1999Go; Lass et al., 1997Go; Tomás et al., 1997Go).

The pregnancy rate after immature oocyte retrieval combined with in-vitro oocyte maturation (IVM) in humans has in general been low. This may partly be explained by the selection of patients, as the majority of these studies have used oocytes that are suboptimal due to the age of the women, endocrinological disturbances or previously failed IVF (Barnes et al., 1996Go; Russell et al., 1997Go; Trounson et al., 1998Go). Recently higher pregnancy rates have been reported in patients with polycystic ovarian syndrome (Cha et al., 2000Go) as well as in regularly menstruating women (Mikkelsen et al., 2000Go). One of the limiting factors in the latter group is the number of oocytes retrieved. In an attempt to establish better selection criteria in patients undergoing IVM, it was examined retrospectively whether the number of retrieved oocytes, the maturation rate and cleavage rate could be predicted in regularly menstruating women with normal ovarian reserve by the use of the following predictive variables on day 3–4: FSH, oestradiol, inhibin B, inhibin A, and the number of ovarian follicles, as these variables have been useful in regular IVF.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Subjects
Data for the study were obtained from 100 consecutive women attending the clinic for IVM treatment due to male factor (n = 48), tubal disease (n = 50) or a combination of these factors (n = 2). In total, 132 aspirations were performed, 18 women underwent two aspirations and seven women underwent three aspirations. The women were equally distributed in both groups of indication (male factor, n = 63; tubal factor, n = 67; combination, n = 2).

All the women had regular menstrual cycles varying from 26 to 35 days and a body mass index between 18 and 29 kg/m2. They had no ovarian abnormalities as assessed by vaginal ultrasound examination and they did not receive any hormone treatment in 3 months before entering the study.

Excluded were all patients with infertility caused by endocrine abnormalities such as hyperprolactinaemia and patients with expected low ovarian reserve (an antral follicle count <3 at 2–5 mm and/or an increased concentration of FSH >15 IU and/or a decreased concentration of inhibin B <45 pg/ml on day 3) (Scott et al., 1989Go; Seifer et al., 1997Go). Also excluded were patients who had previously failed to conceive with conventional IVF for more than three times and patients with possible poor quality of the oocytes which means patients with a low <20% cleavage rate at conventional IVF and women with polycystic ovarian syndrome (PCOS). In the present study PCOS was defined by ultrasound examination (>10 follicles in one plane) and hormone analyses (elevated LH/FSH ratio or elevated androgens).

Transvagianl ultrasound measurements
All transvaginal ultrasound measurements were performed by two observers using a 7.5 MHz transvaginal transducer (B-K Medical A/S, Gentofte, Denmark). The follicular diameter was calculated as the mean of the longest follicular axis and the axis perpendicular to it. The numbers of follicles in both ovaries were added for the antral follicle count. A clinical pregnancy was defined as ultrasonographic evidence of intrauterine fetal heart activity 5 weeks after embryo transfer.

Assays
Immunoreactive FSH concentrations were determined using the commercially available autoanalyser (Immuno I; Bayer A/S, Lyngby, Denmark). The detection limit was 0.1 IU/l and the inter-assay coefficients of variation (CV) were 3% for values below 5.5 IU/ml and 3.2% for values below 15 IU/ml. Oestradiol was measured by the same autoanalyser. The inter-assay CV were 10% for values <100 nmol/l, 8% for values between 200 and 1000 pmol/l, 6% for values between 1 and 2 nmol/l and 5% for values >2 nmol/l respectively. Inhibin A and inhibin B were measured using enzyme-linked immunosorbent assays (Groome et al., 1996Go). The detection limit of inhibin A was 7 pg/ml, and the inter-assay CV were 19.0% for values <14.9 pg/ml, 13.9% for values <31 pg/ml respectively. The detection limit of inhibin B was 20 pg/ml, and the inter-assay CV were 19.0% for values <46 pg/ml, 7% for values between 46 and 238 pg/ml and 6.5% for values between 238 and 634 pg/ml.

Based on previous studies (Evers et al., 1998Go) we used a threshold concentration of 200 pmol/l for oestradiol. No previous studies have used inhibin A as a prognosticator. It was intended to use a threshold limit above the detection limit, and the value 10 pg/ml was chosen.

Experimental design
A transvaginal ultrasound was performed on cycle day 3 and in the case of an ovarian cyst the cycle was cancelled. Based on the total number of follicles (2–5 mm) on day 3 the patients were divided into three groups: the oligofollicular (<5 follicles), the normal (5–15 follicles) and the multifollicular (>15 follicles) ovaries. The second ultrasound examination was performed on day 6–7. Subsequently, ultrasound was performed daily or with an interval of 2–3 days depending on the size of the follicles. Serum concentrations of FSH, LH, oestradiol, progesterone, inhibin A, and inhibin B were obtained on each day ultrasound was performed and on the day of aspiration.

The patients age, number of oocytes retrieved, number of oocytes reaching metaphase II (MII) after 28–36 h, fertilization rate (number of oocytes fertilized divided by number of oocytes reaching MII), cleavage rate (number of oocytes cleaved divided by the number of oocytes fertilized) and clinical pregnancy rate (number of pregnancies with fetal heart activity 5 weeks after embryo transfer divided by number of aspirations) were recorded.

The number of retrieved oocytes was correlated with the basal concentration of FSH, oestradiol, inhibin A and inhibin B respectively. The maturation rate, cleavage rate and pregnancy rate were determined for normal and multifollicular ovaries and for high and low values of oestradiol (threshold 200 pmol/l), FSH (threshold 7.5 IU/l), and inhibin A (threshold 10 pg/ml) respectively.

Oocyte recovery, maturation, fertilization and embryo culture
Oocyte recovery was performed when the size of a leading follicle reached 10 mm and the endometrium had a thickness of at least 5 mm. Oocyte recovery was performed transvaginally with a single lumen needle with a reduced aspiration pressure as described previously (Mikkelsen et al., 1999Go). The follicular aspirates were transferred in tubes to the laboratory and washed on an embryo filter (Falcon 1060) with a pore size of 70 µm to remove erythrocytes and small cellular debris. The retained cells were then resuspended in equilibrated Ham's F-10 (Life Technologies, Roedovre, Denmark) containing both bicarbonate and HEPES buffers and supplemented with 2 mg/ml human serum albumin (HSA; Statens Serum Institute, Copenhagen, Denmark). Oocytes with signs of atresia (dark or shrunken, irregular cytoplasm) or mechanical damage were discarded. Healthy appearing oocytes were matured in tissue culture medium (TCM) 199 (Sigma, Roedovre, Denmark) supplemented with Na pyruvate 0.3 mmol/l, 1500 IU/ml penicillin G, 50 mg/ml streptomycin sulphate, oestradiol 1 µg/ml (all from Sigma), recombinant FSH 0.075IU/ml (Gonal-F; Serono, Geneva, Switzerland), human chorionic gonadotrophin 0.5 IU/ml (Profasi; Serono) and serum from the patient (10%), obtained on the day of aspiration. Oocytes were cultured separately in 25 µl drops of IVM medium under paraffin oil at 37°C in 5% CO2 for 28-36 h (Smith et al., 2000Go).

Oocytes were denuded with hyaluronidase (IVF Science, Göteberg, Sweden) and mechanical pipetting, and the oocyte was classified as having undergone germinal vesicle breakdown when the nuclear membrane was absent and was classified as a mature metaphase II (MII) oocyte when the first polar body was extruded. Motile spermatozoa were prepared by either PurespermTM (Cryos, Copenhagen, Denmark) gradient separation or by swim-up. For intracytoplasmic sperm injection (ICSI), denuded oocytes were placed individually into 5 µl drops of SpermPrep medium (Medi-Cult, Jyllinde, Denmark), and 2 µl of sperm suspension was placed into a 10 µl drop of polyvinylpyrrolidone (IVF Science). Fertilization with ICSI was performed on all MII oocytes. The oocytes were then placed into 10 µl droplets of IVF medium (Medi-Cult) and cultured under oil in Falcon Petri dishes until day 2 or 3 after fertilization. Fertilization was defined as the presence of two pronuclei. A maximum of two embryos were transferred on day 3 after ICSI.

Endometrial priming consisting of 17-ß-oestradiol (Nycomed, Roskilde, Denmark) started on the day of oocyte retrieval. The women received 2 mg orally three times per day. Two days later the luteal phase was supported by vaginal progesterone suppositories (3x100 mg, Progestan, micronized progesterone; Organon, Oss, The Netherlands). Oestrogen and progesterone were continued until pregnancy test, and if pregnancy test was positive they were continued until 50 days gestation.

This study was approved by the local ethics committee, and written informed consent was obtained from all participants.

Statistical methods
Differences in maturation rate, fertilization rate, cleavage rate and clinical pregnancy rate were analysed with Fisher's exact test or {chi}2-test. Because none of the hormone variables were expected to be a normal distribution, the non-parametric Mann-Whitney U-test was used to analyse statistically significant differences between unpaired data.

Pratt's test was used to analyse correlation between parameters. Values were considered significant when P < 0.05. Data are expressed as median and ranges.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
A total of 714 oocytes were aspirated and 509 were judged suitable for IVM (3.8 oocytes per aspiration). In all, 306 (60%) matured to MII and after insemination 223 fertilized (73%) and 193 (87%) cleaved. Fifteen clinical pregnancies were obtained after transfer in 83 cycles, giving a pregnancy rate of 15/132 (11%) per aspiration and 15/83 (18%) per transfer. Twelve healthy babies have been born. One had a cleft palate, the remaining had no malformations. Three pregnancies terminated with an abortion in the eighth to ninth gestational week.

The clinical and hormonal characteristics of the total study group are shown in Table IGo.


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Table I. Hormone values of the study group
 
In 106 cycles the concentrations of oestradiol on day 3 were <200 pmol/l (group 1) and in 26 cycles the oestradiol concentrations were >=200 pmol/l (group 2). The outcome in the groups is shown in Table IIGo. The groups were comparable according to the median age of the patients and the indications for IVM. The number of oocytes, the maturation rate and cleavage rate did not differ between the groups, but significantly more pregnancies were obtained in group 1 (14% per aspiration versus 0% per aspiration, P = 0.03).


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Table II. In-vitro maturation outcome according to oestradiol concentrations on cycle day 3
 
The basal concentrations of inhibin A were available in 103 of the cycles with low basal oestradiol (group 1). To study the potential contribution of the inhibin A concentration on cycle day 3 to predict the IVM outcome, the cycles were subdivided according to their inhibin concentration on day 3. Group 1a: low inhibin A concentration (<10 pg/ml, n = 84 cycles); group 1b: high inhibin A concentration (>=10 pg/ml, n = 19). The outcome for these patients is shown in Table IIIGo. In total 14 pregnancies were obtained in the group with low inhibin A, giving a pregnancy rate of 17% per aspiration.


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Table III. In-vitro maturation outcome according to the concentration of inhibin A on cycle day 3 for those 103 cycles with low concentration of oestradiol (group 1, n = 106 cycles) who had inhibin A available
 
No significant correlation between the basal concentration of inhibin B, inhibin A, oestradiol and the number of oocytes was demonstrated, whereas a negative correlation between the concentration of FSH (P < 0.01) and the number of oocytes was detected. No difference in the quality of the oocytes could be detected, when oocytes from cycles with a low FSH value (<7.5 IU/l) were compared with oocytes from cycles with a higher FSH value (Table IVGo).


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Table IV. In-vitro maturation outcome according to the concentration of FSH on cycle day 3 in 130 cycles with available FSH on day 3
 
More oocytes were obtained from multifollicular ovaries but the pregnancy rate was not improved compared with normal ovaries (Table VGo).


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Table V. In-vitro maturation outcome according to the number of follicles on day 3
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The concentration of oestradiol on cycle day 3 seemed to be useful in predicting the outcome in IVM patients with normal ovarian reserve (basal FSH concentrations <15 IU/l and inhibin B concentrations >45 pg/ml). The group with a low concentration of oestradiol on day 3 (<200 pmol/l) had a significantly higher pregnancy rate compared with the group with a higher concentration (14 versus 0% per aspiration), although the oocyte yield, the maturation rate and cleavage rate did not differ between the two groups. This study also demonstrated that the basal concentration of inhibin A on day 3 may have a putative role as prognostic factor. However, caution is required when interpreting the serum inhibin concentrations from early follicular phase because they are close to the detection limit of the assay.

The current findings parallel previous studies in women undergoing IVF where patients with elevated oestradiol concentrations on cycle day 3 (in a cycle preceding IVF) appeared to have a lower pregnancy rate than the patients with low oestradiol concentrations on day 3 (Evers et al., 1998Go; Scott et al., 1989Go). The concentration of oestradiol is thought to be the critical factor in regulation of FSH in the luteo-follicular phase, and the fall in oestradiol associated with luteolysis is thought to initiate the intercycle rise in FSH. Augmented production of oestradiol or exogenous administration of oestradiol in the early follicular phase may cause a decrease in FSH concentrations and subsequent impaired follicular development (Zeleznek et al., 1981). Although oestradiol is the main trigger of FSH increase in the luteal-follicular transition (Lahlou et al., 1999Go), supraphysiological concentrations of inhibin A have been found to suppress FSH concentration and delay ovulation (Molkness et al., 1996Go). The selection and maturation of oocytes may be impaired in cycles with elevated basal concentrations of oestradiol and inhibin A (Fauser and Van Heusden, 1997Go). The current findings are consistent with this hypothesis.

The full developmental competence of an oocyte and an embryo can be evaluated by live births. There are several studies reporting acceptable maturation, fertilization and embryo cleavage rates after IVM, but only occasional successful pregnancies (Russell et al., 1997Go; Trounson et al., 1998Go). The explanation for low pregnancy rates despite acceptable cleavage rates may be an impaired cytoplasmic maturation or asynchromy between nuclear and cytoplasmic maturation. This may also explain the difference in pregnancy rates observed in the present study between group 1 and 2 despite the similar maturation, fertilization and cleavage rates.

Changes of inhibin A and inhibin B concentration in the menstrual cycle have been described previously (Groome et al., 1996Go; Lockwood et al., 1998Go). Inhibin A remained low in the early follicular phase and increased in the late follicular phase after selection of the dominant follicle. Inhibin A was higher in the luteal phase than the follicular phase and declined synchronously with progesterone and oestradiol during the late lutheal phase (Groome et al., 1996Go). It has been suggested that decline in lutheal phase steroidogenesis and inhibin secretion allowed the rise in FSH which rescued a group of follicles from atresisa.

The serum pattern of inhibin B concentration during menstrual cycle has been shown to be different from that of inhibin A and it is believed that inhibin B is produced by a cohort of recruited follicles. In the present study no correlation was found between inhibin B and the number of oocytes retrieved and, furthermore, the concentration of inhibin B in women with few follicles on day 3 was indistinguishable from the concentration of inhibin B in women with multifolliclular ovaries. This is in accordance with previous observations where no correlation between the number of follicles on day 3 and the concentration of inhibin B on this day has been demonstrated (Lambert-Messerlian et al., 1994Go; Schipper et al., 1998Go). Furthermore, the serum concentrations of day 1–4 inhibin B were not found to be better than age and number of oocytes in predicting the IVF outcome (Corson et al., 1999Go; Hall et al., 1999Go).

Decline in reproductive potential is correlated with the process of follicular depletion and diminished oocyte quality, factors referred to as ovarian reserve (Richardson et al., 1990). Assessment of ovarian reserve is based mainly on measurement of early follicular serum FSH concentration. In IVF treatment, basal FSH concentrations could differentiate between populations with good stimulation characteristics and those with poor characteristics (Muasher et al., 1988Go). When patients had an FSH concentration of >=15 mIU/ml, fewer pregnancies occurred and no pregnancies occurred in patients with FSH >=25 IU/ml (Scott et al., 1989Go). In the current study, the threshold concentration of 15 IU/ml was used for FSH on cycle day 3 and all women with values above this were excluded. Lowering the threshold concentration to 7.5 IU/l did not significantly enhance the pregnancy rate although more oocytes were obtained in cycles with low FSH values.

Although the recovery rate of immature oocytes from unstimulated follicles of 3–9 mm is reduced compared with conventional IVF, the number of follicles on day 3 was shown to predict the number of aspirated oocytes in the present study. Significantly more oocytes were obtained from multifollicular ovaries, compared with normal and inactive ovaries. The developmental potential, however, did not differ between the groups. This corresponds to patients undergoing IVF with ovarian stimulation, where it was found that the number of small follicles was a better predictor of the number of recovered oocytes than ovarian volume alone (Tomás et al., 1997Go).

In conclusion, the concentration of FSH and the number of ovarian follicles counted at ultrasound on day 3 was shown to be valuable in counselling patients before aspiration, due to the risk of a very low number of oocytes aspirated from oligofollicular ovaries. A low oestradiol concentration (<200 pmol/l) on cycle day 3 was a useful prognosticator of pregnancy in IVM. The concentration of inhibin A was of added value (threshold concentration 10 pg/ml), but caution must be taken when interpreting the inhibin A threshold concentrations, because they are close to the detection limit and have a high inter-assay variation. In practice this means that measurement of inhibin A may be less useful in predicting the outcome than measument of oestradiol. The consequences of the application of these criteria need to be examined in the future in a prospective study.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We would like to thank all personnel at the Fertility Clinic of the Institute of Human Reproduction in Copenhagen for their help and technical assistance. The study was in part supported by Medi-Cult A/S.


    Notes
 
3 To whom correspondence should be addressed at: The Fertility Clinic, Institute of Human Reproduction, 3 Fruebjergvej,DK-2100 Copenhagen, Denmark. E-mail: alm{at}ciconia.dk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on October 2, 2000; accepted on February 12, 2001.