1 The Fertility Clinic and 2 Department of Growth and Reproduction, Rigshospitalet, Copenhagen University Hospital, Blegdamsvej 9, 2100 Copenhagen, Denmark
3 To whom correspondence should be addressed at: The Fertility Clinic 4071, Rigshospitalet, Copenhagen University Hospital, Blegdamsvej 9, 2100 Copenhagen. e-mail: drbiba{at}yahoo.com
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Abstract |
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Key words: IVF-ICSI patients/rFSH dosage normogram/ovarian response/standard patients
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Introduction |
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A standard patient is <40 years of age, with two ovaries, a normal serum basal FSH and a regular menstrual cycle. The difficult clinical decision is on the dose to be used for the first week during the first treatment cycle, where the ovarian response to rFSH is basically unknown. From day 8 onwards the response is often evident and adjustments can be made. In subsequent cycles, having assessed the ovarian response in the first cycle, the dosage is far easier.
During recent years data has accumulated, showing that to some extent we are able to predict the ovarian response. The factors which have been investigated as possible predictors include age (Lee et al., 1988; Scott et al., 1989
; Toner et al., 1991
; Rosenwaks et al., 1995
) ovarian volume (Lass et al., 1997
; 1999; Tomas et al., 1997
; Syrop et al., 1999
) number of antral follicles (Chang et al., 1998
; Ng et al., 2000
; Kupesic et al., 2002
), evaluation of ovarian stromal blood flow (Zaidi et al., 1996
; Engmann et al., 1999
; Kupesic et al., 2002
), assessment of the hormonal markers such as serum FSH (Bancsi et al., 2000
;) LH (Noci et al., 1998
), estradiol (E2) (Evers et al., 1998
; Frattarelli et al., 2000
) and inhibin B (Seifer et al., 1997
) as well as cigarette smoking (Van Voorhis et al., 1996
; El-Nemr et al., 1998
).
Although a large number of studies have been conducted in relation to ovarian response a number of methodological problems are encountered including sampling variability and clinical heterogeneity. So far trials have been conducted in relation to single (Scott et al., 1989; Lass et al., 1997
; El-Nemr et al., 1998
; Frattarelli et al., 2000
) or combinations of a few predictive factors (Tomas et al., 1997
; Syrop et al., 1999
; Tinkanen et al., 1999, Ng et al., 2000
). A wide range of sample sizes was present with many small studies (Engmann et al., 1999
; Tinkanen et al., 1999
; Kupesic et al., 2002
).
There are a number of sources of clinical heterogeneity in the studies. Different starting FSH doses were used for controlled ovarian stimulation among the studies from 150 (Bancsi et al., 2002) to 375 IU/day (Engmann et al., 1999
). Within the studies the starting FSH doses were adjusted for age and basal FSH, i.e. not all the patients received the same starting FSH dose. A starting dose of 150 IU/day was given to women <35 years, and 225 IU/day of FSH for older women (Lass et al., 1997
). Both first and second stimulation cycles were included, i.e. the starting FSH dose in the second cycle being determined on the basis of the response to stimulation in the previous cycle. The starting doses from 150 to 375 were given depending on age, previous response to ovarian stimulation and basal FSH level (Engmann et al., 1999
). In other studies women were treated with different stimulation protocols. Additionally, different gonadotrophin preparations were used between and even within the studies.
Among the large number of published studies on ovarian response prediction, only Bancsi et al. (2002), used a consistent methodology including only first cycle patients and administering the same starting FSH dose to all the patients.
The main purpose of the present prospective study was, in a well-defined group of standard patients treated in the first treatment cycle, to examine all the possible predictors of ovarian response and to identify independent predictors through multiple regression analysis. The second aim was to use these findings combined with the data from the literature to suggest an rFSH normogram in order to implement the knowledge clinically.
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Materials and methods |
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Upon the onset of menstrual bleeding in a spontaneous cycle preceding GnRH-analogue treatment patients contacted the clinic. They were seen on day 25 when they received both oral and written information and clinical history was taken. Ovarian ultrasonography was performed using the Panther 2002 ADI (B-K Medical, Gentofte, Denmark) with a 6.5 mHz transvaginal probe (thermal index 1). Exposure time and acoustic output were kept at the lowest level. The number of antral follicles (<5 mm, and <10 mm) was counted. The maximum longitudinal (D1), antero-posterior (D2) and transverse (D3) diameters of each ovary were measured and ovarian volume calculated (D1 x D2 x D3 x 0.523). Ovarian stromal blood flow was evaluated by Power Doppler and a semi-quantitative score was allocated to each ovary according to the number and area of the Power Doppler signals. Score 1 (poor flow) was given in the presence of only a few and scanty signals, suggesting a poor vascularization. Score 3 (good flow) was given in the presence of several pronounced Power Doppler signals. A score of 2 (moderate flow) was allocated to those ovaries with intermediary findings. Total Doppler score (the sum of scores for each ovary) was analysed as a predictive factor, the values being 2, 3, 4, 5 and 6. Blood samples were drawn for assays of: E2, FSH, LH, inhibin B and testosterone. Two investigators were present for each ultrasound examination and video recordings were made.
All patients were treated with the long protocol using nafarelin (Synarela®; Pharmacia, Copenhagen, Denmark) 600 µg/day under down-regulation beginning on day 21 of the cycle and with 400 µg/day from day 1 of rFSH stimulation until the day of hCG. The duration of down regulation was at least 14 days.
After pituitary desensitization was confirmed (i.e. no ovarian cysts, endometrial thickness <5mm) the number of antral follicles was counted (<5 mm, and <10 mm), ovarian volume measured and a Power Doppler score was again allocated for each ovary. Blood samples were taken for serum E2, FSH, LH, and inhibin B. Controlled ovarian hyperstimulation (COH) was commenced with 150 IU/day of rFSH (Puregon®; Organon, Denmark) for the first week of treatment.
On day 8 of stimulation the response was assessed and if considered necessary the dose of rFSH was adjusted accordingly. The dose was increased if the leading follicles were <1011 mm and in case of asynchrony i.e. >4 mm difference between the leading follicle and the next pool. The number of follicles >11, >13, >15 and >17 mm was noted and the ovarian volume was measured. Blood samples were drawn for E2, FSH, LH, inhibin B. The same procedures were repeated on the day of/prior to the administration of hCG (10000 IU, Profasi®, Serono, Denmark).
For the purposes of the study we offered aspiration to all patients who had at least one follicle >17mm on day of/prior to hCG. Aspiration was performed 36 h following hCG administration. The number of follicles aspirated and oocytes retrieved was recorded during aspiration. Standard IVF and ICSI procedures were used. The embryos were transferred on day 2. Four cell embryos with <20 % fragmentation were considered as good quality embryos. All patients were treated with vaginal progesterone (Progestan®, Organon) 200 mg three times a day from the day of embryo transfer until hCG measurement 14 days later.
The study was approved by the regional Ethical Committee of Copenhagen Municipality (KF 01-133/00).
Hormone assays
Blood samples were drawn from antecubal vein and centrifuged. Serum was stored at 20 °C until analysis. Serum E2 was measured by PANTEX (E2)125I kit using the principles of radioimmunoassay. The sensitivity is 10 pg/ml; the intra-assay variation is 4.3%, and inter-assay variation 5.1%. Serum FSH and LH were measured by time-resolved immunofluorometric assay (DELFIA, Wallac, Inc., Turku, Finland), with detection limits of 0.06 and 0.05 IU/l respectively. Intra- and interassay coefficients of variation were both <8% in the FSH and LH assays. Serum inhibin B was measured by a double-antibody enzyme immunometric assay using monoclonal antibody raised against the inhibin B subunit. The detection limit was 20 pg/ml. Intra- and interassay coefficients of variation were 15 and 18% respectively. Testosterone was determined by radioimmunoassay (Count-a-Count: Diagnostic Products, Los Angeles, CA, USA). The detection limit was 0.23nmol/l, and the intra- and inter-assay coefficients of variation were both <10%.
Statistical analysis
The primary end points were the number of aspirated follicles and oocytes retrieved.
Statistical analysis was performed using multiple regression analysis which was carried out in a backward stepwise manner. All of the predictive variables were entered into the model as independent variables to begin with: age, body mass index (BMI), cycle length, smoking status, day 25: total number of antral follicles, total ovarian volume, total power Doppler score, serum E2, FSH, LH, inhibin B and testosterone, the dependent variable being the number of aspirated follicles and retrieved oocytes respectively. All the variables were continuous except for smoking, which was a binary variable (smoker -1, non-smoker -2). The variables were deleted in a backward procedure in order to determine which variables were independent and were needed for the model. Bivariate linear regression analyses were performed with the number of follicles and the number of oocytes as the dependent variables respectively. Significance level for both the multiple and linear regression analyses was 5% (P < 0.05). Statistical analysis of the data was performed with SPSS software (Statistical Package for Social Sciences) for Windows, version 10.0.
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Results |
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Table I shows the clinical and treatment cycle data. The patients age ranged from 25 to 39 years. The age distribution was: 15.2% <30 years, 63.4% between 30 and 35 years of age and 21.4% of the women were >35 years of age. A total of 17.5% had a BMI <20 kg/m2, 59.5% between 20 and 25, and 23% had a BMI >25 kg/m2. Forty-seven women were smokers (32.4%) and 98 (67.6%) were non-smokers.
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Table II gives the results of endocrine screening and ultrasound assessment of the ovaries on day 25 of the menstrual cycle preceding down-regulation.
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The multiple regression analyses results showed that the number of follicles was predicted by: total number of antral follicles, total Doppler score, smoking status, cycle length, serum FSH, LH, E2, testosterone and inhibin B levels. The model given in Table III accounts for 40% of the variability of the number of aspirated follicles (R = 0.647, R2 = 0.418, adjusted R2 = 0.401). The total number of retrieved oocytes was predicted by total number of antral follicles, total Doppler score, smoking status and serum testosterone level. The model in Table IV explained 38% of the variability of the number of retrieved oocytes (R = 0.629, R2 = 0.396, adjusted R2 = 0.379). The total number of antral follicles on the day of baseline ultrasound was the single most significant predictor of both the number of follicles aspirated (P < 0.001) and the number of oocytes retrieved (P < 0.001).
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Bivariate linear regression was performed with the ovarian volume as the independent variable since it was neither in the model with the number of follicles nor the number of oocytes as dependent variables, respectively (Tables V and VI). Ovarian volume in bivariate linear regression was a significant predictor of both the number of follicles (P < 0.001) and the number of oocytes (P < 0.001). It accounted for the 21% of the variability of the number of follicles (R = 0.42, adjusted R2 = 0.21) and for 14% of the variability of the number of oocytes (R = 0.38, adjusted R2 = 0.14). Ovarian volume was significantly correlated to both the number of antral follicles (r = 0.60, P < 0.01) and the total Doppler score (r = 0.42, P < 0.01). This could in turn explain its absence from the multiple regression models for prediction of both the number of follicles and oocytes, i.e. ovarian volume being the confounding variable.
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Suggestion for a dosage normogram
Based on our study we would like to make a suggestion for a simple bed-side FSH dosage score (Table VII) based on the significant parameters of ovarian sonography (antral follicle count, ovarian volume and Power Doppler score) and also on clinical data (age and smoking habits) but not on endocrine tests. The purpose of a normogram would be to prescribe a dose of FSH that was associated with an appropriate number of retrieved oocytes, arbitrarily defined between 5 and 14. The cut-off values for each variable in the normogram were drawn from our own study population and were defined in relation to our definition of appropriate ovarian response.
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The normogram was constructed with the intention of achieving these dosages. The weight of each parameter was based on the most significant and clinically well-established variables (antral follicle count and ovarian volume) given the highest scores, whereas the less significant variables were given the lowest scores. The Doppler score was attributed a score in between. We opted to give it a lower score as this has only been shown in the present study.
It has to be stressed that this FSH dosage normogram is a construction based on scientifically documented variables but additionally on clinical assumptions.
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Discussion |
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Our independent predictors of the number of oocytes as assessed by the multiple regression analysis could account for 38% of the variation of ovarian response in comparison with 25% in the study by Tinkanen et al. (1999) and 27% in the work by Ng et al. (2000
).
The total number of antral follicles on day 25 of a spontaneous cycle was an independent predictor of both the number of follicles and oocytes. This finding is in agreement with the results of a number of investigations (Chang et al., 1998; Bancsi et al., 2002
).
With the improvements of the ultrasound equipment colour Doppler sonography (Zaidi et al., 1996; Engmann et al., 1999
) and Power Doppler (Kupesic et al., 2002
) have been used to assess ovarian circulation in order to predict the ovarian response to COH. While the frequency-based colour Doppler sonography provides directional information of the flow within the vessel, Power Doppler sonography is monochromatic, it does not provide directional information but its sensitivity to low flow makes it more useful in studying ovarian perfusion. It is also less angle-dependent and not susceptible to aliasing (Meyerowitz et al., 1996
; Pairleitner et al., 1999
).
In the present study we assessed ovarian blood flow using a semi-quantitative Power Doppler score (2D). We showed that the total power Doppler score was an independent predictor of both the number of follicles and oocytes. This is a new finding. The technique is easy to perform and it is not time consuming. It is however difficult to make strict criteria for quantification. The 3D Power Doppler indices (Pairleitner et al, 1999; Kupesic et al., 2002
) may help to overcome this problem, although it is fair to say that presently they have not gained wide acceptance in clinical use.
Interesting and new data emerged from our analyses regarding the cycle length. Within the regularly cycling women study population (cycle length 2135 days), it was an independent predictor of the number of aspirated follicles but not the retrieved oocytes. Women with short cycles had less aspirated follicles compared with women with longer cycles. This is in agreement with the fact that there is a gradual decline in cycle length with the ovarian ageing as shown by Treolar et al. (1967) and Münster et al. (1992
) in a large cohort of regularly cycling women.
Smoking has been associated with an earlier menopause and impaired ovarian function by a number of large epidemiological studies (Jick et al., 1977; Adena et al., 1982
; McKinlay et al., 1985
). The validity of self-reporting smoking is often questioned but a meta-analysis of published studies comparing self-reported smoking status with biochemical validation suggested generally high levels of sensitivity (87%) and specificity (89%) for self-report (Patrick et al., 1994
). Our data have confirmed that actual smoking status is a predictor of both the number of follicles and the number of oocytes.
Age was a significant predictor of both the number of follicles (P = 0.03) and the number of oocytes (P = 0.03) in bivariate linear regression analyses. Interestingly, however, age was not an independent predictor in our study. This is in contrast with other studies (Ng et al., 2000) but may be explained by the inclusion of several other factors that are related to the biological age of the ovaries.
The importance of basal FSH levels remains controversial although it is well accepted that the patients with elevated basal FSH level exhibit diminished ovarian responsiveness. Our study shows that even in patients with normal basal FSH levels, FSH levels and the number of aspirated follicles were inversely related. Similar findings regard the basal E2 levels as shown by Frattarelli et al. (2000) where patients with elevated E2 levels were more likely to respond poorly to gonadotrophins. Although inhibin B was an independent predictor of the number of follicles, it accounted for only 3% of the variability of both the number of follicles and oocytes when analysed separately in bivariate linear regressions. The fact that our patient population was restricted to women with normal basal FSH levels, may account for this observation.
How do we implement the knowledge we have on predictive factors in clinical practice?
The problem is that standard patients treated with standard doses frequently do not exhibit standard responses. The question is how, and to what extent, it is possible to avoid the inappropriate responses by administering appropriate dose of rFSH. The variability of the responses may be due to inherent biological mechanisms in relation to differences in the number of recruitable follicles, follicle sensitivity to rFSH and pharmacodynamics. On the other hand they may also be due to factors that may be predicted and at least partly controlled.
On the basis of our data but also taking into account clinical experience and safety considerations we would like to suggest an rFSH dosage normogram composed of the following variables: total number of antral follicles day 25, total Doppler score day 25, total ovarian volume day 25, age and smoking status (Table VII). This normogram is based on clinical and ultrasound data and doesnt include endocrine parameters.
Several suggestions for a normogram could be made. Our intention was to design a simple normogram, which would allow an easy individual approach to the determination of rFSH starting dose by taking a history and ultrasonography. The primary focus in the construction of the normogram was the number of oocytes and we therefore included the variables from the oocyte model.
Volume of the ovaries is an indirect marker of the ovarian responsiveness. The number of follicles was a better predictor than the ovarian volume and it may be used as a first method for predicting the ovarian response to gonadotrophins (Tomas et al., 1997). This is consistent with our findings as the number of antral follicles on day 25 independently predicted the number of both the follicles and oocytes, whereas the volume did not. However, ovarian volumetry by transvaginal sonography is accurate and easily performed in most women with very small intra- and interobserver variations (Goswamy et al., 1988
; Higgins et al., 1990
; Lass et al., 1997
). In the situation where the quality of the ultrasound image may be impaired by the ovarian localization, obesity or other factors estimation of the antral follicle number may prove to be a difficult task especially the very small antral follicles with 23 mm in diameter. Ovarian volume could thus be considered as a safety variable.
Although age did not prove to be an independent predictor in the multiple regression it was significantly correlated to both the number of oocytes and follicles in the bivariate regressions. We have included age in the dosage normogram because evidence has accumulated over the years, which has shown that fertility declines with age (Lee et al., 1988; Scott et al., 1989
; Toner et al., 1991
; Faddy and Gosden, 1995
; Rosenwaks et al., 1995
).
One might criticise the normogram for not including any endocrine parameters although we have them in the model for both oocytes and follicles (Tables III and IV). Hormonal determinations may have inter-cycle variability, are expensive and are not commonly used in standard patients, at least in Scandinavia.
The rFSH dosage normogram is easy to use in a clinical setting. To illustrate the use of the normogram a 36-year-old woman, non-smoker, with 10 antral follicles, ovarian volume of 8 ml, and a Doppler score 4 should be given 220 IU/day of rFSH (20 + 0 + 90 + 90 + 20). A 28 year old woman, smoking 5 cigarettes per day, with 30 antral follicles, ovarian volume of 11ml and Doppler score 6 should be given 120 IU/day of rFSH (0 + 10 + 50 + 60 + 0).
The normogram has not been experimentally tested but a randomized trial comparing a standard dose of 150 IU/day of rFSH versus a normogram defined individual dose is presently under way.
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References |
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Submitted on August 16, 2002; resubmitted on December 5, 2002; accepted on January 8, 2003.