OVULATION INDUCTION. Hsiang, Kaohsiung, Taiwan (FAX: ; adm.cgmh.org.tw).

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1 OVULATION INDUCTION Significantly superior response in the right ovary compared with the left ovary after stimulation with follicle-stimulating hormone in a pituitary down-regulation regimen Kuo-Chung Lan, M.D., a,b Fu-Jen Huang, M.D., a Yi-Chi Lin, B.S., a Fu-Tsai Kung, M.D., a Ting-Hsun Lan, M.D., c and Shiuh Young Chang, M.D. d a Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital, Kaohsiung Medical Center, Chang Gung University College of Medicine, Niaosung Hsiang; b Graduate Institute of Clinical Medical Sciences, Chang Gung University; c Department of Prosthodontics, Kaohsiung Medical University Hospital; and d Sheng An Hospital, Kaohsiung, Taiwan Objective: To compare the differences between the right and left ovarian responses in patients undergoing standard in vitro fertilization (IVF) treatment. Design: Prospective, observational study. Setting: A university hospital in Taiwan. Patient(s): Seven hundred eighty-nine consecutive patients undergoing IVF or intracytoplasmic sperm injection. Intervention(s): None. Main Outcome Measure(s): The number of oocytes retrieved, fertilization rates, number of grade 1 zygotes and number of embryos produced, embryo arrest-free rate, and mean number of top-quality embryos on the day of transfer. Result(s): The number of follicles R16 mm in diameter on day 2.5 versus 2.2 of human chorionic gonadotropin (hcg) administration, the number of follicles R10 mm in diameter on 6.5 versus 6.1, the number of oocytes (3.6 vs. 3.0), the percentage fertilization (79.9 vs. 74.6%), and the mean number of mature oocytes and grade 1 zygotes (2.3 vs. 1.8 and 1.79 vs. 0.93, respectively), and the mean available number of top-quality embryos on transfer day 1.39 versus 0.73 were all statistically significantly greater in the right compared with the left ovary; however, the pregnancy and implantation rates were similar. Conclusion(s): There are statistically significant differences between the right and left ovarian responses in IVF patients with healthy ovaries, and the right ovarian responses are superior to the left ovarian responses. (Fertil Steril Ò 2010;93: Ó2010 by American Society for Reproductive Medicine.) Key Words: IVF, ICSI, ovulation induction, ovarian response Since the first in vitro fertilization (IVF) pregnancy was delivered in 1978, this procedure has resulted in thousands of pregnancies and opened a vast new frontier of research and treatment for the infertile couple. Over the past 30 years, the benefits of ovarian stimulation have transformed the field of reproductive endocrinology as it pertains to the treatment of infertility. Pregnancy rates with IVF improve as the number of high quality embryos available for transfer increases; Received June 20, 2008; revised November 19, 2008; accepted December 22, 2008; published online February 9, K-C.L. has nothing to disclose. F-J.H. has nothing to disclose. Y-C.L. has nothing to disclose. F-T.K. has nothing to disclose. T-H.L. has nothing to disclose. S.Y.C. has nothing to disclose. Supported by grant NMRPG from the National Science Council of Taiwan. Reprint requests: Kuo-Chung Lan, M.D., Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital, Kaohsiung Medical Center, Chang Gung University College of Medicine, 123 Ta-Pei Road, Niaosung Hsiang, Kaohsiung, Taiwan (FAX: ; blue@ adm.cgmh.org.tw). therefore, ovarian stimulation agents to produce multiple oocytes for IVF are advantageous. In addition, ovarian stimulation has improved IVF outcomes by enabling the development of multiple embryos and a choice of best embryos for transfer (1). Ovarian stimulation rests on the concept that increased circulating follicle-stimulating hormone (FSH) levels sustain the growth of additional cohort follicles by raising the intrafollicular FSH above a critical threshold, thereby rescuing the follicles from atresia. This rescuing of additional follicles from the cohort is referred to as recruitment. The size of the recruitable cohort is proportional to the ovarian reserve of the follicles (2). Ovarian stimulation initiated before day 5 overrides dominant follicle selection, resulting in multifolliclular development. Both the magnitude and duration of the FSH dose determine the ongoing multifollicular development. Basic science findings show that, even though the right and left ovaries are histologically and embryologically similar, /10/$36.00 Fertility and Sterility â Vol. 93, No. 7, May 1, doi: /j.fertnstert Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.

2 differences do exist between venous drainage, anatomic relationships, and cyclical physiologic control between the two ovaries. The basic endocrine control is the same for both ovaries, but there are discrete interovarian and intraovarian physiologic differences controlling both follicular development and side of ovulation in each cycle (3). Potashnik et al. (4) made the surprising observation that in women with two healthy ovaries, ovulation takes place significantly more often from the right ovary than the left ovary. Fukuda et al. (5) also observed ovulation from the right ovary more frequently than the left ovary. Furthermore, oocytes from the right ovary lead to the establishment of pregnancy more often than oocytes originating from the left ovary; the pattern is the same in fertile and infertile women (5). Whether the right or left ovaries respond similarly to the artificial situation of ovulation induction or superovulation cycles has been less thoroughly assessed. Although comparisons have been made between the right and left ovarian responses during IVF for patients with a unilateral diseased or absent ovary (6), or compared pooling of unilateral ovarian responses (3), no study has examined the paired response of bilaterally normal ovaries to gonadotropin stimulation within the same individual patient. We therefore determined if there were any significant differences between right and left ovarian responses in patients with healthy ovaries undergoing standard IVF treatment. MATERIALS AND METHODS Patients We performed a prospective observational study involving 789 patients undergoing consecutive IVF or intracytoplasmic sperm injection (ICSI) cycles since January 1, All the couples completed the standard infertility workup, which included hysterosalpingography and hormone assays in women and semen analysis in men. Patients were unselected for age, sperm parameters, or infertility criteria. The inclusion criteria were the presence of both ovaries, no history of ovarian surgery, and no ovarian abnormalities as assessed by transvaginal ultrasound. Exclusion criteria were the presence of ovarian cysts and inaccessible ovaries and cycles for oocyte donation. The laboratory facilities, clinical strategy, and protocol for controlled ovarian hyperstimulation followed the standard down-regulation regimen we published previously elsewhere (7, 8). A single team of embryologists coordinated all procedures, thereby ensuring that both the culture protocols and embryo assessments were standardized. This study was approved by the ethics committee of Chang Gung Memorial Hospital. Approval from the institutional review board was obtained for analysis of this series. scored according to the Z-score system. The system took account of nuclear size and alignment, and nucleoli number and distribution. We used G1.2 medium (Scandinavian IVF Science. Gothenburg, Sweden) for culture of embryos on days 1 to 3, and G2.2 medium (Scandinavian IVF Science) was used for culture of embryos from days 3 to 5 or 6. On day 3, all of the transferable embryos were assessed for blastomere number and regularity, and presence and volume of cytoplasmic fragmentation was performed. After 2 days of culture in G2.2 medium, blastocyst formation was evaluated. The scoring assessment for blastocysts was based on the expansion state of the blastocyst and on the consistency of the inner cell mass and trophectoderm cells. The Z1 zygotes had equal numbers of nucleoli aligned at the pronuclear junction (the absolute number was not counted, but was between three and seven). The top-quality embryos were considered grade 1 day-3 embryo morphologies (eight cells, blastomeres of equal size, and no cytoplasmic fragments) or day-5 blastocysts (full blastocysts onward; the development of the inner cell mass with numerous, tightly packed cells; the trophoectoderm with many cells forming a cohesive epithelium). Establishment and Follow-Up of Pregnancy In our program, we have routinely offered blastocyst transfer to patients with >3 eight-cell embryos on day 3. Luteal phase supplementation of micronized progesterone (Utrogestan, 800 mg intravaginally daily; Piette International Laboratories, Drogenbos, Belgium) was begun on the day of oocyte retrieval, and 5000 IU of human chorionic gonadotropin (hcg) was administered on day 6 after oocyte recovery in all patients. Pregnancy was confirmed by detecting hcg in the urine 2 weeks after transfer. Clinical pregnancy was determined by identifying a gestational sac at 7 weeks gestation by means of transvaginal ultrasonography. If conception had occurred, micronized progesterone supplementation was provided for an additional 4 weeks. Statistical Analysis The Sigmastat statistical package (Jandel Corporation, San Rafael, CA) was used for data analysis. Continuous data are summarized as the mean standard deviation (SD). As this was an observational rather than intervention study, we included 789 cycles to maximize the power of the study. The ovarian response study included the paired Student s t-test comparison of the means. The embryo transfer study included the Mann-Whitney rank-sum test for comparison of means, and the Fisher s exact test for proportions. All P values are two-sided, and P<.05 was considered statistically significant. Assessment of Fertilization, Embryo Culture, and Zygote and Embryo Grading After the ICSI procedure, oocytes were cultured as in the standard IVF procedure and assessed for the presence of pronuclei after 16 to 18 hours of incubation. The zygotes were RESULTS For the 789 cases included in this study, general characteristics related to the causes of infertility, maternal age, assisted reproduction technique (IVF or ICSI), and outcome were noted and are presented in Table 1. The comparison of the 2270 Lan et al. Ovarian response in IVF patients Vol. 93, No. 7, May 1, 2010

3 TABLE 1 Demographic characteristics of 789 cycles that underwent IVF/ICSI. Characteristic Value Number of cycles with 789 at least one oocyte retrieved Number of cycles that 493 underwent IVF Number of cycles that 296 underwent ICSI Number of cycles 763 transferred Age of female (20 45) partners (years) Body mass index ( ) Infertility Primary 475 Secondary 314 Duration of infertility (1 18) (years) Number of oocytes (1 28) retrieved Endometrial thickness on day of hcg (cm) Estradiol (pg/ml) on ( ) Progesterone (ng/ml) on Number of indications Tubal factor 172 Male 264 Endometriosis 96 Ovulatory factor 88 Unexplained and 48 others Combined factors 121 Normal fertilization 79.5% (4145/5213) rate Number of mean (1 5) embryos transferred Clinical pregnancy 42.9% (328/763) rate/transfer cycle Implantation rate 20.3% (379/1863) right versus left ovarian response to controlled ovarian hyperstimulation is shown in Table 2. The number of follicles R16 mm in diameter on 2.5 versus 2.2, the number of follicles R10 mm in diameter on 6.5 versus 6.1, the number of oocytes (3.6 vs. 3.0), the percentage fertilization (79.9% vs. 74.6%), the mean number of mature oocytes and grade 1 zygotes (2.3 vs. 1.8 and 1.79 vs. 0.93, respectively), and the mean available number of top-quality embryos transferred on day 1.39 versus 0.73 were all statistically significantly different on the right versus left. However, as Table 3 reveals, the pregnancy and implantation rates were similar in the embryo transfer model from the right or left ovary only. DISCUSSION It has been a matter of debate whether ovulation occurs from alternating sides, from the same side, or randomly. There is some evolutionary basis for thinking that the right and left ovaries may differ. Similar to other species, the mechanism in human by which the two ovaries differ in their activities is unknown. Assuming that the two ovaries experience an equal endocrine control of pituitary hormones, it is worthy of note that the midluteal serum estradiol and testosterone levels were higher in right-sided ovulation than left sided in a previous study (5). This difference could be related to anatomic asymmetry or be the result of different gonadotropin exposure time due to stasis of flow. Although both ovaries receive arterial blood supply via the ovarian arteries directly from the aorta, the venous drainage differs as the right ovarian vein drains directly to the inferior vena cava whereas the left first drains to the left renal vein then to the inferior vena cava (3, 9). Balakier and Stronell (10), using the perifollicular peak velocity and resistance index values, suggested that the right ovary is more active for most of the reproductive years and the left ovary dominates in the later years. To evaluate whether the vascularization is different, we are presently performing color Doppler ultrasound measurements. However, this does not exclude the possibility that right-side ovary, for some unknown reason, possesses an intrinsically enhanced response potential that causes the observed effects. Fukuda et al. (11) reported that ovulation switching from the left to the right ovary in two successive cycles may increase the chances of pregnancy during intrauterine insemination and/or IVF natural cycles. The underlying mechanism of an enhanced implantation potential of embryos derived from the right ovary is also not clear. The total number of retrieved oocytes decreases with increasing age and increasing ovarian volume, pituitary desensitization using leuprolide acetate has no effect on overall ovarian volume measurements, and the number of small antral follicles is also unchanged after pituitary desensitization (12). It is reasonable that normal ovaries recruit a significantly higher number of follicles and yield a significantly higher number of oocytes compared with contralateral ovaries that had undergone cystectomy surgery (13). The difference between right or left ovaries in natural cycles or ovulation induction, especially in pituitary down-regulation regimens, is interesting. There is no predilection of one ovary Fertility and Sterility â 2271

4 TABLE 2 Comparison of right and left ovarian response to controlled ovarian hyperstimulation in 789 cycles. Right ovary Left ovary P value (0 13) (0 21) <.001 of oocytes collected (0 10) (0 8) <.001 of mature oocytes Follicles R10 mm in diameter on (1 21) (1 23).005 Follicles R16 mm in diameter on (0 12) (0 15) <.001 Normal fertilization rate 79.9% 74.6% < (0 11) (0 7) <.001 of Z1 zygotes Embryo arrest-free rate 66.3% 52.5% <.001 Mean available number of top-quality embryos on transferred day (0 8) (0 4) <.001 over the other with a gonadotropin-releasing hormone/human menopausal gonadotropin (GnRH/HMG) ovulation induction protocol for IVF (6). In the aforementioned ovarian surgery model, it also was reported that no significant differences were identified between the left and right ovaries in the number of follicles recruited and oocytes obtained from each ovary in the normal control patients (13). Thomson et al. (3) performed a prospective, case-controlled study in 200 consecutive IVF cycles and concluded that there were no statistically significant differences between the right and left ovarian responses in IVF patients with healthy ovaries. Our results differ from the three previous reports in that there was a statistically significantly superior response in the right ovary compared with the left after stimulation with FSH in a pituitary down-regulation regimen. We attribute the difference to the study model design, and in particular the statistical analysis method. If two samples are paired (i.e., if each person is used as his or her own control or if the sample consists of different people who are matched on a one-to-one basis), then the paired t-test is appropriate (14). To this end, we constructed paired Student s t-tests to analyze the paired response of normal bilateral ovaries to gonadotropin stimulation within the same individual patient. The pregnancy and implantation rates were similar in the embryo transfer model from the right or left ovary only in our study. Admittedly, the embryo transfer model is deficient and difficult to draw definite conclusions from. But it is consistent with our earlier study (8); it revealed that embryo characteristics resulting in availability of embryos were major factors contributing to successful pregnancies. Embryos derived from the right ovary were not the sole factor for pregnancy. TABLE 3 Pregnancy rates after embryo transfer. Embryos from right ovary only (n [ 316) Embryos from left ovary only (n [ 124) P value s of embryos transfer Implantation rates 25.3% (178/704) 20.1% (51/254).103 Pregnancy rates 40.8% (129/316) 37% (46/124) Lan et al. Ovarian response in IVF patients Vol. 93, No. 7, May 1, 2010

5 We conclude that there are statistically statistical differences between the right and left ovarian responses in IVF patients with healthy ovaries. The right ovarian response is superior to the left ovarian response in follicle recruitment and oocyte retrieval. Our study may provide biological information for consultation for IVF ovarian response, but it would not change clinical practice in any way. REFERENCES 1. Jennings JC, Moreland K, Peterson CM. In vitro fertilisation. A review of drug therapy and clinical management. Drugs 1996;52: Fauser BC, Van Heusden AM. Manipulation of human ovarian function: physiological concepts and clinical consequences. Endocr Rev 1997;18: Thomson AJ, Gazvani MR, Wood SJ, Meacock SC, Lewis-Jones DI, Kingsland CR. Comparison of ovarian response in right and left ovaries in IVF patients. Hum Reprod 2001;16: Potashnik G, Insler V, Meizner I, Sternberg M. Frequency, sequence, and side of ovulation in women menstruating normally. Br Med J (Clin Res Ed) 1987;294: Fukuda M, Fukuda K, Andersen CY, Byskov AG. Right-sided ovulation favours pregnancy more than left-sided ovulation. Hum Reprod 2000;15: Lass A, Croucher C, Lawrie H, Margara R, Winston RM. Right or left ovary which one is better? Hum Reprod 1997;12: Lan KC, Huang FJ, Lin YC, Kung FT, Chang SY. Zona-free versus laser zona-assisted hatching blastocyst transfer: a comparison of outcomes. Fertil Steril. Published online May 17, Lan KC, Huang FJ, Lin YC, Kung FT, Hsieh CH, Huang HW, et al. The predictive value of using a combined Z-score and day 3 embryo morphology score in the assessment of embryo survival on day 5. Hum Reprod 2003;18: Last RG. Anatomy: regional and applied. 7th ed. Edinburgh: Churchill Livingstone, 1984: Balakier H, Stronell RD. Color Doppler assessment of folliculogenesis in in vitro fertilization patients. Fertil Steril 1994;62: Fukuda M, Fukuda K, Andersen CY, Byskov AG. Ovulation jumping from the left to the right ovary in two successive cycles may increase the chances of pregnancy during intrauterine insemination and/or in vitro fertilization natural cycles. Fertil Steril 2006;85: Sharara FI, Lim J, McClamrock HD. The effect of pituitary desensitization on ovarian volume measurements prior to in-vitro fertilization. Hum Reprod 1999;14: Nargund G, Cheng WC, Parsons J. The impact of ovarian cystectomy on ovarian response to stimulation during in-vitro fertilization cycles. Hum Reprod 1996;11: Rosner B. Fundamentals of biostatistics. 5th ed. New York: Duxbury Press, 2000: Fertility and Sterility â 2273