Pii: s0015-0282(02)04574-0

FERTILITY AND STERILITY௡
Copyright 2003 American Society for Reproductive Medicine Printed on acid-free paper in U.S.A.
Coordination of early antral follicles by
luteal estradiol administration provides a
basis for alternative controlled ovarian
hyperstimulation regimens
Renato Fanchin, M.D.,a Joa˜o Sabino Cunha-Filho, M.D.,a Luca Maria Schona¨uer, M.D.,aIsaac Jacques Kadoch, M.D.,a Paul Cohen-Bacri, M.D.,b and Rene´ Frydman, M.D.a Hoˆpital Antoine Be´cle`re, Clamart, and Laboratoire d’Eylau, Paris, France Objective: To investigate whether luteal E administration reduces size discrepancies of early antral follicles.
Design: Prospective, crossover study.
Setting: ART unit, Clamart, France.
Patient(s): Sixty women and 120 cycles.
Intervention(s): On cycle day 3 (baseline day 3), all women underwent measurements of early antral follicles
by ultrasound and serum FSH and ovarian hormones. From day 20 until the next cycle day 2, 30 of them
received oral 17␤-E , whereas the remaining women served as controls. The day after E discontinuation (E
day 3) or on subsequent cycle day 3 (control day 3), participants were reevaluated as on baseline day 3.
Main Outcome Measure(s): Magnitude of follicular size discrepancies.
Result(s): Follicular size discrepancies and follicular diameters were significantly attenuated on E day 3 (3.7
Ϯ 0.5 mm) as compared with baseline day 3 (4.9 Ϯ 1.0 mm), but not in controls (5.0 Ϯ 0.8 vs. 4.9 Ϯ 0.8 mm).
FSH (4.3 Ϯ 1.9 vs. 7.3 Ϯ 3.3 mIU/mL) and inhibin B (34 Ϯ 28 vs. 71 Ϯ 32 pg/mL) levels were consistentlylower on E day 3 than on baseline day 3 but remained unchanged in controls.
Conclusion(s): Luteal E administration reduces the size and improves the homogeneity of early antral
follicles on day 3. This approach may be instrumental in synchronizing follicular development duringcontrolled ovarian hyperstimulation. (Fertil Steril௡ 2003;79:316 –21. 2003 by American Society for Repro-ductive Medicine.) Key Words: FSH, estradiol, early antral follicles, controlled ovarian hyperstimulation
The foremost objective of controlled ovar- dissimilar sizes, which range from 2 to 8 mm in ian hyperstimulation (COH) is to ensure the adequate functional and morphologic matura- both of these interrelated phenomena may be tion of early antral follicles to increase the instrumental in the establishment of the follic- Fanchin, M.D., Departmentof Obstetrics and number of viable oocytes and the probability of ular dominance. Indeed, although it is conceiv- conception. One of the key points of such a able that complex intrafollicular mechanisms procedure is the achievement of adequate syn- concur to determine follicular sensitivity to Hoˆpital Antoine Be´cle`re,157 Rue de la Porte de chronization of follicular growth so that ovu- lation can be triggered when most follicles larger follicles are more responsive to this hor- have reached concomitant maturation. How- 4980; E-mail: renato.
fanchin@abc.ap-hop- ever, to attain satisfactory follicular coordina- mechanisms underlying the heterogeneity of tion during COH, the physiological heteroge- early antral follicles sizes during the early fol- neity of selectable follicles observed during the early follicular phase should probably be over- A possible explanation for this phenomenon involves the exposure of early antral follicles to During the first days of the follicular phase gradient FSH levels during the late luteal in the menstrual cycle, early antral follicles are phase. During the ultimate phase of the men- 0015-0282/03/$30.00PII S0015-0282(02)04574-0 discrepantly sensitive to FSH and exhibit strual cycle, up to 5 days before the onset of Study protocol. Horizontal black bars represent menstrual bleeding. On baseline/day 3, E /day 3, and control/day 3, hormonal and ultrasound measurements were performed.
Fanchin. Coordination of early antral follicles. Fertil Steril 2003. menstrual bleeding paralleling the corpus luteum clearance, or excretion; [4] body mass indexes ranging from demise, FSH levels increase progressively to preserve antral 18 to 27 kg/m2; [5] no current hormone therapy; [6] adequate follicles from atresia and ensure subsequent growth visualization of ovaries in transvaginal ultrasound scans.
According to their inherent sensitivity to FSH, it is possible Informed consent was obtained from all women, and this that some early antral follicles are able to respond to lower investigation received the approval of our internal Institu- amounts of FSH than others and, therefore, to start their tional Review Board. For personal reasons (n ϭ 4) or major development during the late luteal phase This premature, protocol violation (n ϭ 2), six women did not complete the gradual exposure of follicles to FSH may accelerate the two subsequent observation cycles required by the protocol development of more sensitive follicles and accentuate size and had to be excluded from the analysis. Therefore, the discrepancies observed during the first days of the subse- population studied was limited to 60 participants undergoing Hence, we decided to challenge the hypothesis that size discrepancies among early antral follicles result, at least in Study Protocol
part, from their early and progressive exposure to FSH. For The study protocol is summarized in On day 3 this we elected to artificially lower endogenous FSH secre- of their menstrual cycles (baseline/day 3), all 60 women tion during the luteal phase by administering E and underwent blood sampling for serum FSH, inhibin B, and E to measure follicular and hormonal profiles on day 3 in two measurements at approximately 9:00 A.M. Later in the morn- consecutive menstrual cycles that were either preceded or ing, ultrasound scans of their ovaries were performed. Sub- not preceded by luteal E administration.
sequently, women were randomized to receive luteal E2treatment or to serve as controls. As shown in MATERIALS AND METHODS
participants who were included in the E -treated group (n ϭ Subjects
30) received micronized 17␤-E oral tablets (4 mg/day; We prospectively studied 66 female volunteers, 20 – 41 Provame`s, Cassenne Laboratories, Puteaux, France) in the years of age. All participants met the following inclusion evening at 8:00 P.M. from day 20 of the same cycle until day criteria: [1] regular, ovulatory menstrual cycles every 25–35 2 of their next cycle. We chose the 4 mg/day dose for E p.o.
days; [2] both ovaries present; [3] no current or past diseases administration because of its reported efficacy in reducing affecting ovaries or gonadotropin or sex steroid secretion, endogenous FSH and in preventing early follicular growth FERTILITY & STERILITY௡
Participants who were included in the control group (n Statistics
The measure of central tendency used was the mean and the measure of the variability was the SD. Because of the pairwise design of this study, data from each participant on women (E /day 3) or on day 3 of the subsequent cycle in E day 3 or on control day 3 were compared with corre- control women (control/day 3), hormonal and ultrasound sponding data for the same participant on baseline day 3 by measurements similar to the preceding cycle (baseline/day 3) using the paired Student’s t-test. To evaluate the magnitude were performed. In addition, participants were asked to of follicular size discrepancies from baseline day 3 to E day compute their baseline and subsequent menstrual cycle 3 and from baseline day 3 to control day 3, we tested the lengths and to report possible subjective changes in men- homogeneity of variances by using the Levene test for equal strual bleeding characteristics. In E -treated women, compli- variances This test is less sensitive than F-tests to ance of treatment was monitored to detect any protocol departures from normality and allows a comparison of the dispersion of data around the mean independent of meanvalues. In addition, SD:mean ratios for follicular sizes were Ultrasound Measurements
also calculated. The present crossover study was powered to Ultrasound scans were performed using a 4.5–7.2 MHz detect anticipated differences of 0.5 mm for follicular sizes multifrequency transvaginal probe (Siemens Elegra, Sie- and 2 mm for ovarian volume calculation at Ͼ80% power at mens S.A.S., Saint-Denis, France) by one single operator a 0.5 significance level. PϽ.05 was considered statistically (J.S.C.F.), who was not aware of the treatment schedule or the hormonal results. The objective of the ultrasound exam-inations was to evaluate the number and sizes of early antralfollicles and to calculate the mean ovarian volume. We considered all follicles that measured 2–12 mm in meandiameter (mean of 2 orthogonal diameters). The choice of Ultrasound Results
not excluding follicles measuring 9 –12 mm was based on Follicular and ovarian measurement results are summa- the fact that, according to our own experience, some women rized in As expected, the number of antral follicles display marked acceleration of follicular development.
did not change significantly from one cycle to another inE -treated women and in controls. In contrast, we observed In an attempt to optimize the reliability of ovarian follic- a significant reduction of mean follicular sizes in E -treated ular assessment, the ultrasound scanner that was used was women from baseline/day 3 to E /day 3 but not in controls.
equipped with a tissue harmonic imaging system which In agreement with this, mean ovarian volume decreased allowed improved image resolution and adequate recogni- significantly in women treated with E and remained un- tion of follicular borders. Ovarian volumes, calculated ac- changed in controls. In addition, we observed a remarkable cording to the formula for an ellipsoid (0.526 ϫ length ϫ attenuation of follicular size discrepancies on E day 3 as height ϫ width) were the mean volume for both ova- compared with baseline day 3 (PϽ.0001). This was not ries. Intra-analysis coefficients of variation (CVs) for follic- noticed between baseline day 3 and control day 3. Consis- ular and ovarian measurements were Ͻ5%, and their lower tently, SD:mean ratios for follicular sizes were significantly lower on E /day 3 than on baseline/day 3 but not on control/ day 3 as compared with baseline/day 3, which confirms the Hormonal Measurements
improvement in follicular size homogeneity observed in All blood samples were obtained by venipuncture, and E -treated women. Incidentally, it is noteworthy that ultra- serum was separated and frozen in aliquots at Ϫ20°C for sound measurements made on baseline/day 3 were strictly subsequent centralized analysis. Serum FSH was measured similar in women included in the E -treated and control by an immunometric technique using an Amerlite kit (Ortho Clinical Diagnostics, Strasbourg, France). Intra-assay andinterassay CVs were, respectively, 5% and 7%, and lower Hormonal Results
limit detection was 0.1 mIU/mL for FSH. Serum inhibin B Hormonal results are also presented in In women was determined by double antibody enzyme-linked immu- who were administered E during the luteal phase, serum nosorbent assay (Serotec, Varilhes, France) as described inhibin B levels were significantly lower on E /day 3 as elsewhere Lower limit detection was 10 pg/mL, and compared with baseline day 3, whereas no significant lon- intra-assay and interassay CVs were Ͻ6% and Ͻ9%, respec- gitudinal change in inhibin B levels was noted in controls.
tively, for inhibin B. Serum E was determined by an im- As expected, E administration raised serum E to levels munoometric technique using an Estradiol-60 Amerlite kit comparable to those observed during the late follicular phase (Ortho Clinical Diagnostics). Lower limit detection was 14 of the menstrual cycle (114 Ϯ 57 pg/mL on E day 3). Serum pg/mL, and intra-assay and interassay CVs were 8% and 9%, E levels did not vary significantly in controls from baseline/ day 3 to control/day 3. Administration of E lowered serum Fanchin et al.
Ultrasound and hormonal results during two consecutive menstrual cycles in women receiving E during the luteal Fanchin. Coordination of early antral follicles. Fertil Steril 2003. FSH levels on E /day 3 as compared with baseline/day 3.
stringent methodological parameters such as population se- However, in women who did not receive E treatment, serum lection, randomized crossover design, accurate ultrasound FSH levels remained steady from one cycle to the other. CVs technology, and independent data analysis.
for FSH from the first to the second cycle were significantly Our results indicated that E administration during the higher in E -treated women than in controls (40% vs. 17%, luteal phase effectively reduced both the size discrepancies PϽ.01). As for ultrasound measurements, hormonal results and the mean diameter of early antral follicles, consistent obtained on baseline/day 3 were closely similar in women with a decrease in mean ovarian volume. The observation of receiving E as compared with those who served as controls.
a subtle but significant lengthening of menstrual cycle dura- Clinical Results
tion in women pretreated with E is in keeping with this As anticipated, the E -treated and the control groups were effect. Indeed, although ovulation has not been monitored in comparable with regard to ages of women (33.3 Ϯ 0.6 vs.
the present study, menstrual cycle lengthening after E treat- 33.3 Ϯ 0.5 years) and body mass index (21.8 Ϯ 0.4 and 21.8 ment may be attributed to a longer growth course of smaller Ϯ 0.3 kg/m2). A significant lengthening of mean menstrual antral follicles to ovulation. Furthermore, these results are in cycle duration was observed in participants receiving E accordance with data from previous experiments conducted (29.4 Ϯ 1.3 days, PϽ.0001) as compared with their baseline cycles (27.8 Ϯ 1.2 days). This phenomenon was not ob- Earlier studies have shown that administration of physi- served in controls (28.0 Ϯ 1.1 vs. 27.9 Ϯ 0.8 days, respec- ological E doses effectively prevents and postpones the tively). Moreover, E treatment did not alter the baseline intercycle FSH elevation In line with this, the cycle length in E -treated patients (27.8 Ϯ 1.2 days) as present investigation showed lower FSH levels (Ͻ4.5 mIU/ compared with controls (27.9 Ϯ 0.8 days). Participants did mL) on the day after E discontinuation as compared with not indicate any significant change in their menstrual bleed- baseline (Ͼ7 mIU/mL). This phenomenon may not be at- ing characteristics in E -treated as compared with baseline tributable to physiological intercycle FSH fluctuations be- cause it was not duplicated in the control group. In addition,the intercycle CV for FSH observed in E -treated patients DISCUSSION
(40%) significantly exceeded the intercycle CVs in controls The present study was designed to investigate whether E (17%) and the CVs for FSH measured in the early follicular administration during the luteal phase could affect the degree phase as reported by other investigators (Ͻ18%) of development of early antral follicles during the first days Therefore, both the reduction of follicular size and heter- of the subsequent follicular phase. We wanted to challenge ogeneity observed by the present study lead us to hypothe- the hypothesis that developmental asynchrony of early antral size that, after E administration, serum FSH levels might follicles possibly results from the gradual FSH elevation that have remained beneath the putative FSH threshold for early occurs during the late luteal phase. This phenomenon may antral follicular development Indeed, during the late promote asynchronous growth of follicles because of their luteal phase of the menstrual cycle, spontaneous demise of intrinsic dissimilar sensitivity to FSH Hence, the corpus luteum leads to a progressive rise in FSH that through its putative suppressive effect on FSH secretion starts approximately 5 days before menses and reaches se- E administration might attenuate follicular size rum levels Ͼ7 mIU/mL as early as 3 days before the onset heterogeneity. To address this issue, our investigation set of menstrual bleeding It is possible that the obtained FERTILITY & STERILITY௡
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