Maximizing outcomes for poor responders to controlled ovarian hyperstimulation (COH) in IVF: the use of microdose flare GnRH agonist induction to gonadotropin stimulation in women with previous IVF failure.

compared to 51.6% in the motile sperm group (P ⬍ 0.005) and embryos were available for replacement in the motile sperm group in 76.9% of the cycles compared to 50% of the cycles in the non-motile sperm group (P ⬍ 0.05). The clinical pregnancy rate was 12.5% in the non-motile group compared to 12.8% in the motile group (P ⫽ 0.9742) and the delivery/ ongoing pregnancy rate was 6.25% in the non-motile sperm group compared to 7.69% in the motile sperm group (P ⫽ 0.8516). Conclusions: ICSI performed with totally non-motile testicular spermatozoa selected by the modified hypo-osmotic swelling test results in comparable pregnancy rates ICSI using motile testicular spermatozoa, in cases of non-obstructive azoospermia despite the fact that the fertilization and cleavage rates are lower in the non-motile sperm group. Supported by: Alexandria Fertility Centre.

P-360 Comparison of two methods for assessment of seminal oxidative stress in infertile men. R. A. Saleh, N. Esfandiari, S. S. Al-Dujaily, R. K. Sharma, A. J. Thomas, A. Agarwal. The Cleveland Clinic Foundation, Cleveland, OH. Objective: Seminal oxidative stress (OS) is defined as an imbalance between reactive oxygen species (ROS) production and total antioxidant capacity (TAC), and is highly correlated with male infertility. Evaluation of seminal OS status is of important diagnostic and prognostic value in a male infertility clinic. The objective of this study was to compare two methods for evaluation of the seminal OS: 1) by measurement of ROS directly in neat semen, and 2) by measurement of ROS in washed semen, TAC in seminal plasma, and the calculation of a composite ROS-TAC score. Design: Prospective study in a male infertility clinic. Materials/Methods: Semen samples were obtained from 9 normal donors and from 34 infertile men. Donors were selected on the basis of normal semen parameters and negative Endtz (ⱕ1 ⫻ 106 WBCs/mL) as per World Health Organization guidelines. Levels of ROS were determined in neat (unprocessed) semen and after simple wash and re-suspension, by a chemiluminescence assay using luminol as a probe. Results were expressed as ⫻104 counted photons per minute/20 ⫻ 106 sperm/mL. Patients were classified as OS-negative (ⱕ1.5 ⫻ 104 cpm) and OS-positive (1.5 ⫻ 104) based on ROS levels in neat semen of normal donors. Total non-enzymatic antioxidant capacity (TAC) was measured in seminal plasma by an enhanced chemiluminescence assay and results were expressed as trolox equivalents. A composite value of ROS-TAC score was calculated by principal component analysis using ROS values in washed semen. Results: Median (25%, 75% inter-quartile range) values of ROS in neat, and washed semen, TAC and ROS-TAC score of donors and the 2 patient groups are shown in the table. ROS levels in neat semen was highly correlated with ROS in washed semen (r ⫽ 0.87, P ⬍ 0.001), and with ROS-TAC score (r ⫽ ⫺0.75, P ⬍ 0.001). Donors (n ⫽ 9)

Variable

OS-negative (n ⫽ 11)

OS-positive (n ⫽ 23)

ROS-neat semen 0.3 (0.2, 0.9) 0.3 (0.2, 1) 19 (6, 143) (⫻ 104 cpm) ROS-washed semen 10 (4, 17) 79 (13, 177) 1468 (514, 11496) (⫻ 104 cpm) TAC (trolox 908 (736, 1129) 797 (575, 966) 739 (627, 1047) equivalents) ROS-TAC score 53 (51, 55) 49 (47, 52) 36 (32, 44) Variable 4

ROS-neat semen (⫻ 10 cpm) ROS-washed semen (⫻ 104 cpm) TAC (trolox equivalents) ROS-TAC score

a

b

c

0.9 0.03 0.31 0.04

0.0001 0.0003 0.24 0.001

⬍0.0001 0.0008 0.94 0.001

a: Donors vs. OS-negative patients; b: Donors vs. OS-positive patients; c: OS-negative patients vs. OS-positive patients. Results were analyzed by Wilcoxon Rank-Sum Test, P ⬍ 0.05 was significant. Conclusions: Our results demonstrate a strong relationship between ROS levels in neat semen and the composite value of ROS-TAC score from

FERTILITY & STERILITY威

washed semen. We conclude that ROS measurement in neat semen can be incorporated as a routine andrology laboratory procedure for assessment of oxidative stress. Supported by: A grant from The Cleveland Clinic Foundation.

P-361 In vitro maturation of germinal vesicle oocytes. P. G. Papaioannidou, A. Borini, S. Garetti, M. A. Bonu, C. Flamigni. Univ of Bologna, Bologna, Italy. Objective: Germinal Vesicle (GV) oocytes, which are aspirated during the pick up, cannot mature in a few hours and if they are inseminated they cannot be fertilized. Even after in vitro maturation of GV oocytes, the fertilization rate, the developmental potential of the resulting embryos and pregnancy rate is extremely low. So, GV oocytes, which are collected during the pick-up, are not used routinely for in vitro fertilization (IVF). As the success of IVF depends on the number of the aspirated oocytes, we decided to mature in vitro the GV oocytes and use them for IVF, in order to increase the success rate of assisted reproduction. Design: Follicular Fluid (FF), taken from a big follicle that contained a mature oocyte, was added to the culture medium to promote in vitro maturation of GV oocytes. The study was approved by the local Institutional Review Board. Materials/Methods: 50 GV oocytes, which were collected during follicular aspiration after ovulation induction, were cultured for 30 hours in modified Tissue Culture Medium 199 (M199, Sigma, USA) containing 30% FF. FF of a big follicle containing a mature oocyte was collected during oocyte aspiration, was centrifuged at 300 g for 10 min and the supernatant was filtered through a 0.22 micro-meter filter. FF of each patient was used for maturation of her own oocytes only. After in vitro maturation, the resulting Metaphase II (MII) oocytes were microinjected. Normal fertilization was assessed and the resulting embryos, if of a good quality, were transferred or cryopreserved. Only oocytes from ICSI cycles were used for the study. The Student’s t-test was used for statistical analysis. Results: Germinal Vesicle Breakdown (GVBD) occurred in 41 out of 50 oocytes (82%) and 34 of these oocytes progressed to the stage of MII (83%) with a final maturation rate of 68%. ICSI was performed in 33 oocytes (97%) and 22 of them (67%) were fertilized; but normal fertilization with the presence of 2pn occurred in 16 cases (fertilization rate 49%). All the fertilized oocytes cleaved (cleavage rate 100%) and gave embryos of equal quality (or sometimes better) to the embryos derived from mature oocytes. 11 out of 16 embryos (69%) were of high quality (grade 1 or 2) and 9 embryos (56%) were used either for transfer (4 embryos) or for cryopreservation (5 embryos). A twin pregnancy was achieved after transfer of two embryos one of which was MI and the other GV during the pick-up, with one gestational sac corresponding to the embryo derived from the GV oocyte. Conclusions: In vitro maturation of GV oocytes with the addition of FF in the culture medium seems to give acceptable results in terms of maturation and fertilization rates. Although the percentage of the resulting embryos (32%) was not very high, more than 50% of these embryos could be used either for transfer or cryopreservation. In some cases, the embryos derived from the GV oocytes were of better quality than those derived from the MII. There were also cases in which transfer was possible only by utilising embryos derived from GV oocytes. It is also interesting that cleavage rate was 100%. These results show that GV oocytes can mature in vitro, can be fertilized and the resulting embryos can implant and give a pregnancy. In vitro maturation of GV oocytes with the use of FF in the culture medium seems thus to be an additional simple and cost effective method to increase the number of oocytes and finally to increase the success rate in assisted reproduction.

ART: OVARIAN STIMULATION P-362 Maximizing outcomes for poor responders to controlled ovarian hyperstimulation (COH) in IVF: the use of microdose flare GnRH agonist induction to gonadotropin stimulation in women with previous IVF failure. A. G. Mark, E. S. Ginsburg, K. V. Jackson, B. W. Walsh, C.

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Racowsky. Brigham and Women’s Hosp, Harvard Medical Sch, Boston, MA. Objective: Poor responders frequently receive minidose or microflare regimens in addition to sweetening. The efficacy of these stimulation regimens remains unclear. This study was designed to address this issue. Design: Retrospective review of 1,393 IVF or ICSI cycles performed between August 98 and January 01. Materials/Methods: Poor responders (⬍42 yo) were identified who failed to conceive with their first ART cycle using either the minidose or microflare protocols and then proceeded to a second cycle of minidose or microflare, with or without sweetening (n ⫽ 55). The relative efficacies of the four stimulation regimens were assessed. The minidose protocol utilized 0.5 mg lupron on CD 21 with cessation of lupron and start of gonadotropins on CD2 (n ⫽ 19). The microflare protocol utilized OCP’s until CD1, then 0.05 mg lupron bid with gonadotropins on CD2 (n ⫽ 36). Outcome measures included number of eggs retrieved, E2: follicle ratio, % fertilization, % good quality embryos (ⱖ8 cells with ⬍10% fragmentation), implantation rate per embryo transferred, and ongoing/delivered rate per cycle start. Results were analyzed using Chi Square with Yates correction or Mann U Whitney tests where relevant with p ⬍ 0.05 considered significant. Results: The results are summarized in Table 1. Table 1.

Cycle Starts Age Day 3 FSH E2:Follicle # of Eggs %2pn/MII Egg %ⱖ8 cells, ⬍10% frag %Implanted (fetus) %Ongoing-Delivered/ start

Minidose/ FSH

Minidose/ hMG/FSH

Microflare/ FSH

Microflare/ hMG/FSH P-value

9 38.0 ⫾ 4.0 7.7 ⫾ 4.1 126 ⫾ 43 11.3 ⫾ 8.5 68.1 ⫾ 32.9 5.9 (3/51)

10 37.2 ⫾ 4.0 7.8 ⫾ 2.5 124 ⫾ 53 14.9 ⫾ 5.2 56.3 ⫾ 14.4 7.1 (4/56)

13 36.7 ⫾ 3.1 9.0 ⫾ 4.4 215 ⫾ 90 6.8 ⫾ 3.6 53.5 ⫾ 35.3 26.3 (10/38)

23 37.8 ⫾ 3.5 9.2 ⫾ 2.7 196 ⫾ 55 8.1 ⫾ 4.1 73.8 ⫾ 21.0 31.3 (31/99)

4.3 11.1

0 0

6.9 15.4

16.9 39.1

0.63 0.34 0.002 0.008 0.181 0.0001 0.028 0.050*

* All minidose versus all microflare cycles p ⫽ 0.041.

Conclusions: Regardless of sweetening, microflare was associated with significantly improved outcomes compared with minidose. Pregnancy rates were doubled in microflare cycles with sweetening. Furthermore, embryo quality was significantly improved, potentially accounting for the improved ongoing-delivered rates. Therefore, the microflare GnRH agonist protocol should be used with sweetening in poor responders failing to conceive in a minidose or microflare cycle. P-363 Day six estradiol level predicts cycle cancellation in GnRHa flare IVF cycles. P. Kovacs, B. R. Witt. Albert Einstein Coll of Medicine, Bronx, NY. Objective: The purpose of this study is to assess the predictive value of day 6 estradiol level (“flare effect”) for cycle outcome and to compare the response to miniflare and standard flare protocols in IVF-ET cycles. Design: Retrospective analysis of IVF-ET cycles during 1999 –2000 where flare protocols were used. Materials/Methods: 71 IVF-ET cycles were identified. 60 patients were poor responders (identified based on previous cancelled cycle or elevated baseline FSH level (9 IU/ml)), 11 had other indications. The cycles were analyzed as 2 groups. Group A (n ⫽ 38) (“miniflare”): GnRHa (Lupron) 40 mcg sc. bid from day 2 with FSH started on day 4. Group B (n ⫽ 33) (“standard flare”): Lupron 1 mg on day 2 and 3, then 0.5 mg from day 4, with FSH from day 2. Both groups were pretreated with OCPs for at least 21 days. Serum E2 and ultrasound (U/S) monitoring were initiated on day 6 of cycle. After day 6 U/S and blood tests were performed daily or every other day. Cycles with inappropriate E2 rise or with ⬍3 mature follicles were cancelled. When at least two follicles were ⬎17 mm 10,000 U HCG im was given and transvaginal oocyte retrieval was performed 34 hours later. Embryo transfer was done on day 3. 12 days following ET a serum ␤hCG was done to establish pregnancy. Luteal phase was supported with im

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Abstracts

progesterone in oil. Data was collected for cycle parameters and outcome. Statistical analysis was performed using t-test, chi-square, and ROC analysis. Results: ROC analysis showed that day 6 E2 level was predictive of cycle cancellation with a cut-off of 75 pg/ml (E2ⱕ75 pg/ml, 15/26 [57.7%] vs E275 pg/ml, 7/45 [15.5%], p ⬍ 0.0001). The same cut-off value was not predictive of pregnancy outcome. The two protocols were compared among poor responders (Group A: n ⫽ 36, Group B: n ⫽ 24). The two groups had similar baseline characteristics (age, FSH) and cycle parameters (peak E2, number of oocytes retrieved, cycle length, number of embryos, quality of embryos). Group B patients required less mean (⫾ SEM) number of ampules of FSH (64.7 ⫾ 8.1 vs 89.5 ⫾ 6.4). There was a tendency for higher cancellation rate (16/36 [44.4%] vs 7/24 [29.2%] and lower pregnancy rate (2/36 [5.6%] vs 5/24 [20.8%]) in group A, however neither difference reached statistical significance. Day 6 E2 ⱕ75 pg/ml remained predictive of cycle cancellation but not for pregnancy outcome. Conclusions: A day 6 E2 level ⬍75 pg/ml (regardless of flare protocol) was predictive of cycle cancellation. Early cancellation should be considered at that time when a flare effect is absent. There is a tendency for fewer cycle cancellations and higher pregnancy rates with the “standard flare.” P-364 Lifetime high day 2 FSH and diagnostic accuracy in IVF. I. S. Tummon, I. Hardy, M. Lee, V. R. Cardone, M. Seibel, M. Summers. Fertility Ctr of New England, Winchester, MA; Fertility Ctr of New England, Reading, MA; Fertility Ctr of New England, Dedham, MA. Objective: To test diagnostic accuracy of lifetime high day 2 FSH in an unrestricted IVF population. Design: Retrospective analysis of lifetime high day 2 FSH with outcome measure: birth or ongoing pregnancy. Materials/Methods: 2,058 day 2 FSH measurements from 539 women aged 38 ⫾ 4 years (mean ⫾ SD) were studied. FSH standardization was WHO 2nd International Standard (IRP 78/549). Immunoassay was performed using Technicon Immuno 1 (Bayer, Tarrytown NY) (normal follicular phase range less than 9.6 mIU/mL). 730 consecutive IVF stimulations (lifetime IVF cycle 3 ⫾ 2) were begun with flare GnRH-agonist/gonadotropin or clomiphene/gonadotropin/⫾GnRH-antagonist. All diagnoses were included. Prior to stimulation 80 cycles were deferred on account of elevated FSH (20 ⫾ 7 mIU/mL). 81 cycles were not started on account of functional cysts, persistent luteal function or elevated estradiol. Results: Lifetime high day 2 FSH was greater than 10 mIU/mL in 40% of 730 cycles. Examined in increments of 2 mIU/mL for lifetime high FSH, confidence intervals for crude birth/ongoing pregnancy rates were not different from 4 –16 mIU/mL. Receiver operator characteristic curves for cycle and lifetime high FSH had areas of 0.5, indicative of poor test accuracy. FSH greater than 16 mIU/mL gave positive likelihood ratios of 2.6 for cycle FSH (fair test) and 1.9 (poor to fair) for lifetime high FSH. With respect to outcome as the dependent variable, neither cycle FSH, nor lifetime high FSH, nor number of elevated FSHs, nor proportion of FSHs that were elevated were significant independent variables by multiple regression analysis. After starting treatment, 72 cycles (10%) were cancelled for poor response. Lifetime high FSH was greater in cancelled cycles 14.7 ⫾ 8 versus 9.8 ⫾ 4 in cycles completed to oocyte retrieval, (p ⫽ 0.001) and risk of cancellation was related to lifetime high FSH, (F ⫽ 74, r2 ⫽ 0.09, p ⫽ 0.001).

Vol. 76, No. 3, Suppl. 1, September 2001