Fertility and Sterility - Mauro Schimberni

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Use of a medium buffered with n-hydroxyethylpiperazine-n-ethanesulfonate (HEPES) in intracytoplasmic sperm injection procedures is detrimental to the outcome of in vitro fertlization Francesco Morgia, B.S.,a Monica Torti, B.S.,a Monica Montigiani, B.S.,a Claudio Piscitelli, M.D.,a Annalise Giallonardo, M.D.,a Mauro Schimberni, M.D.,a Pierluigi Giannini, M.D.,a and Marco Sbracia, M.D.b a

Bioroma Centro di Riproduzione Assistita Casa di Cura “Paideia” and b Center of Endocrinology and Reproductive Medicine, Rome, Italy

Objective: This study was conducted to determine whether N-hydroxyethylpiperazine-N-ethanesulfonate (HEPES)– buffered medium used for the microinjection of sperm into oocytes may be detrimental for the embryo. Design: Controlled randomized study. Setting: Private IVF center. Patient(s): Women (n ⫽ 708) undergoing ICSI. Intervention(s): The women were randomized into two study groups: 2,204 oocytes from 357 women were treated using a medium buffered with bicarbonate without HEPES during the ICSI procedure, and 2,168 oocytes from 351 women were treated using a medium buffered with HEPES during the ICSI procedure. Main Outcome Measure(s): Fertilization rate, degeneration rate, triploid rate, cleavage rate, embryo quality, pregnancy rate, implantation rate, and abortion rate. Result(s): Oocytes treated with a HEPES-buffered medium showed a statistically significant higher rate of triploid and degenerated oocytes after fertilization with ICSI compared with oocytes treated with a medium without HEPES. The embryos obtained from oocytes microinjected with a HEPES-buffered medium showed a statistically significant higher rate of highly fragmented embryos compared with the controls. Pregnancy rate and implantation rate were statistically significantly lower in the patient group with oocytes treated with the HEPES-buffered medium. The other parameters evaluated did not show any statistically significant differences. Concluxion(s): Our study showed that the use of media buffered with HEPES, during the microinjection of sperm into the oocytes, is detrimental for IVF outcome and should be avoided. (Fertil Steril威 2006;xx:xxx. ©2006 by American Society for Reproductive Medicine.) Key Words: HEPES, intracytoplasmic sperm injection, IVF outcome, embryo quality

The widespread use of assisted reproductive techniques has highlighted the role of the culture media, which have to allow embryos to develop in the correct way for 3– 6 days before replacement in the uterus.

promote cell fragmentation in the early cleavage stage embryo (5). For these reasons the media generally used in IVF for human embryos are manufactured without HEPES and phosphate buffers (3, 4, 15).

In recent years several media have been developed to allow human embryos to arrive at the blastocyst stage (1– 4). In these media phosphate buffer and organic buffer, such as N-hydroxyethylpiperazine-N-ethanesulfonate (HEPES), are avoided, because several reports have shown that these buffers may be toxic or detrimental to embryo development (5–14).

In the microinjection of sperm into the oocyte during intracytoplasmic sperm injection (ICSI), sperm selection is generally performed using a medium buffered with HEPES (16). Long standing time of gametes under the microscope for the procedure may change the pH of the medium, and the presence of an organic pH buffer may help to maintain the pH in a stable state.

Early reports showed that bicarbonate is essential for fertilization and embryo development, whereas phosphate buffer and organic buffer are not as efficient as bicarbonate (8 –12). Furthermore, it has been shown that HEPES may

However, the use of these substances may be detrimental to embryo development after fertilization, especially with the direct injection of an organic buffer into the cytoplasm of oocytes as shown in an early report (17). In that study the authors reported that the HEPES injected into the oocyte determined a rise in intracellular pH with a reversible meiotic arrest.

Received June 23, 2005; revised and accepted October 25, 2005. Reprint requests: Marco Sbracia, M.D., Center for Endocrinology and Reproductive Medicine, Via Carlo Porta 10, 00153, Rome, Italy (FAX: 39-06-5880096; E-mail: [email protected]).

0015-0282/06/$32.00 doi:10.1016/j.fertnstert.2005.10.050

For these reasons we hypothesized that, because the ICSI procedure can be performed in a few minutes by experienced

Fertility and Sterility姞 Vol. xx, No. x, Month 2006 Copyright ©2006 American Society for Reproductive Medicine, Published by Elsevier Inc.

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embryologists, avoiding HEPES in the medium used for microinjection may improve the IVF outcome. In this study we tested with a controlled trial whether avoiding the use of a medium buffered with HEPES during ICSI may be useful for embryo development after fertilization. MATERIALS AND METHODS All patients referred to Bioroma, Rome, Italy, undergoing their first ICSI cycle from January 2002 to December 2003 were eligible for the study. The study was reviewed and approved by the institutional review board. The patients were randomly assigned by a computergenerated number sequence to the two arms of the study: one in which a medium buffered only with bicarbonate, not containing HEPES (IVF; Vitrolife, Kungsbacka, Sweden), was used for the ICSI procedure and other procedures, including sperm washing and oocyte collection, and the other in which the medium with HEPES (Gamete; Vitrolife) was used during ICSI. A total of 357 cycles were included in the group treated without HEPES, and 351 in the control group with HEPES. For patients attending more than one cycle of IVF, only the first cycle was included in the study. Each patient was included in only one arm of the study. Azoospermic patients were excluded from the study. Controlled ovarian hyperstimulation was performed according to the long protocol in all patients. Subcutaneous buserelin, 0.4 mg daily, on days 22 to 24 of their previous cycle was administered to the patients. When suppression was confirmed by E2 and ultrasound examinations, FSH was commenced at 300 IU daily (Gonal-f; Serono, Rome, Italy) on the second day of the menstrual cycle. From the seventh day of stimulation in both groups, daily monitoring of follicle size by ultrasound was performed, and plasma levels of E2 were measured. From this stage the dose of FSH was adjusted depending on the individual response of each patient. The criteria used for triggering ovulation with 10,000 IU hCG (Gonasi HP 5000, AMSA, Rome, Italy) IM were plasma E2 between 1,000 and 4,500 pg/mL and at least four follicles ⬎16 mm diameter. The cycle was canceled in case of poor ovarian response, when less than three follicles were observed on the ninth day, or in case of ovarian hyperstimulation syndrome, E2 ⬎4,500 pg/mL. Oocyte retrieval was performed under ultrasound control by the transvaginal route on day 0, 36 h after the injection of hCG. ICSI was performed in all cases according to published procedures (16). Oocytes were observed 18 h after ICSI for their pronuclei. The embryologists performing ICSI were blind to the solution used. The embryos obtained were categorized on day 3 into three categories, depending on their morphologic appear2

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ance. Grade A had 6 – 8 or more equal and regular blastomeres without the presence of cytoplasm fragments; grade B had less than 6 – 8 unequal blastomeres with or without cytoplasmatic fragments; and grade C were fragmented (more than 50%) embryos (18). The embryos were transfered 72 h after insemination using the Wallace embryo transfer catheter (H. G. Wallace, UK). All transfer procedures were performed by the same physician to avoid inter-operator variability. All pregnancies were confirmed by a rising titer of serum ␤-hCG from 12 days after embryo transfer and ultrasound demonstration of the gestation sac 4 weeks after the transfer. Biochemical pregnancies alone have not been included. The same luteal phase support was used in both groups: 50 mg daily progesterone (Prontogest; AMSA) intramuscularly from the day of replacement. Statistical Analysis All statistical analyses were performed using the SPSS statistical package (SPSS, Chicago, IL). Pregnancy rate and implantation rate were the primary outcomes, and secondary outcomes were days of stimulation, E2 at the day of hCG, number of oocytes collected, fertilization rate, degeneration rate, number of triploid embryos, number of embryos obtained, quality of embryos, number of embryos transfered, and abortion rate. Mann-Whitney U test and Student t test were used for continuous variables (days of stimulation, E2 at the day of hCG, amount of FSH administered, number of oocytes collected, number of embryos obtained, and number of embryos transfered), and Fisher exact test was used for dichotomous variables (clinical pregnancy rate, implantation rate, fertilization rate, quality of embryos, degeneration rate, number of triploid embryos, and abortion rate). A sample size analysis a priori was done for a differencece of 20% between the two groups in pregnancy rate. The statistical results of P obtained were corrected for the number of comparisons performed. RESULTS In Table 1 the epidemiologic data of the two patient groups are reported. No statistically significant differences were found between the two study groups. The two groups were homogeneous for female age, male age, time of infertility, and frequency of infertility causes. In Table 2 the clinical data of controlled ovarian hyperstimulation are reported. No statistically significant differences were found between the two study groups for days of stimulation, E2 levels at the hCG day, FSH amount administered to the women, number of oocytes recovered, metaphase two rate, or immature oocytes rate. Vol. xx, No. x, Month 2006

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TABLE 1 Epidemiological data of patients included in the study groups. Without HEPES

Control

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the control group (37.8% vs. 28.2%; P⬍.01; 18.3% vs. 12.3%; P⬍.01). No differences were observed for the number of embryos transfered, top-quality embryo rate transfered, or abortion rate between the two groups. The data are reported in Table 3.

P

No. of patients 357 351 — Women’s age (y) 35.4 ⫾ 4.2 36.1 ⫾ 4.1 NS Men’s age (y) 38.2 ⫾ 4.5 37.8 ⫾ 4.3 NS Time of infertility (y) 3.3 ⫾ 1.3 3.1 ⫾ 1.1 NS Causes of infertility: Ovulatory 16.2% 15.7% NS Tubal 24.9% 25.1% NS Endometriosis 19.6% 18.8% NS Male 34.2% 34.5% NS Other 5.0% 5.9% NS Please Supply Citation Line. Fertil Steril 2006.

In the group of 357 patients without HEPES, ICSI was performed on 2,204 oocytes and normal fertilization was observed in 69.9.%, and in the control group of 351 patients ICSI was performed on 2,168 oocytes with a normal fertilization rate of 69.2%; no statistically significant differences were found. The degenerated oocytes rate after ICSI was 6.5% in the group without HEPES and 8.8% in the control group (P⬍.007). Triploid embryo rate was 3.3% in the group without HEPES and 6.1% in the control group (P⬍.0001). No differences were observed for embryo cleavage rate between the two groups. The frequency of bad-quality embryos with high fragmentation was statistically significantly lower in the group without HEPES, 30.2%, compared to the control group, 37.5% (P⬍.0001). The embryos graded B were statistically significantly higher in the group without HEPES, 46.3%, compared to controls, 39.4% (P⬍.001). The rate of top-quality embryos obtained and replaced in uterus were similar in both groups. The pregnancy rate and implantation rate in the group without HEPES were statistically significantly higher than in

DISCUSSION In recent years the culture media for IVF have been improved to respond to the needs of human embryos during the first stages of development. With the introduction of sequential media, each step from fecundation to the blastocyst embryo stage development has a specifically designed medium without a phosphate buffer and with only bicarbonate buffer and different concentrations of pyruvate lactate and glucose (1– 4). In the ICSI procedure, to avoid an excessive pH change during micromanipulation under the microscope, a medium buffered with HEPES, an organic pH buffer, is generally used. However, it is well known that this organic pH buffer may be detrimental to the zygote and during early embryo cleavage stages and may be toxic to embryo development (5–14). An early report showed that the injection of HEPES into starfish eggs inhibited meiosis reinitiation (17). It has been shown that a concentration of 25 mmol/L HEPES in the culture medium is detrimental to embryo development in bovines (19). Several authors showed the detrimental role of HEPES in the media used for fertilization (8, 9, 11). Furthermore, the role of HEPES in promoting embryo fragmentation and retarded development has also been reported (5, 20 –22). The ICSI procedure was introduced in 1991 (16), and now experienced embryologists can perform it in a reduced time, avoiding the oocytes’ prolonged exposure to light and a different environment from the incubator. For these reasons we have evaluated the possibility of using a medium without HEPES during the ICSI procedure. The data of our study showed that the use of a medium without HEPES during the ICSI procedure permits a statis-

TABLE 2 Clinical data of controlled ovarian hyperstimulation.

No. of cycles Day of stimulation FSH administered (IU) E2 hCG/day levels (pg/mL) No. oocytes harvested No. of M2 oocytes No. of immature oocytes

Without HEPES

Control

P

357 12.7 ⫾ 2.5 3,427 ⫾ 2,085 2,575 ⫾ 1,425 8.0 ⫾ 4.9 87.0% 13.0%

351 13.1 ⫾ 2.4 3,510 ⫾ 1,897 2,579 ⫾ 1,085 8.2 ⫾ 4.8 86.8% 13.2%

— NS NS NS NS NS NS

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TABLE 3 Results of comparison between ICSI cycles using during microinjection medium with HEPES and without.

No. of oocytes treated No. of oocytes fertilized Triploid Degenerated No. of embryos cleaving (%) Embryos obtained Embryo grading: Grade A (%) Grade B (%) Grade C (%) Embryos transfered Top-quality embryo Pregnancy rate Implantation rate Abortion rate

Without HEPES

Control

P

2,204 69.9% 3.3% 6.5% 68.1% 4.2 ⫾ 2.7

2,168 69.2% 6.1% 8.8% 65.1% 4.0 ⫾ 3.0

— NS .0001 .007 NS NS

348 (23.4) 688 (46.3) 450 (30.2) 2.5 ⫾ 0.8 36.0% 37.8% 18.3% 14.8%

323 (23.0) 553 (39.4) 525 (37.5) 2.6 ⫾ 0.9 33.8% 28.2% 12.3% 15.1%

NS .001 .0001 NS NS .01 .01 NS

Please Supply Citation Line. Fertil Steril 2006.

tically significant increase of the pregnancy rate compared to oocytes fertilized in a medium containing HEPES, as well as a statistically significant increase of the implantation rate in the same group. Furthermore, our study clearly showed that the use of HEPES in the media for microinjection is detrimental for human embryos, especially when it is directly injected into the oocyte, where it increased the amount of degenerated oocytes, triploid embryos, and embryo fragmentation. All our results are consistent, because in each step we observed an improvement when HEPES was avoided in the medium for microinjection: from fertilization, with the reduction of degenerated oocytes and triploid zygotes, to the eight-cell embryo stage, with the reduction of highly fragmented embryos, and after embryo replacement, with the increase of pregnancy and implantation rate, even though the embryos transfered in the two groups were similar for number and quality. The detrimental role of HEPES in embryo development and IVF outcomes observed in our study may be due to the increase of intracellular pH caused by this organic buffer when injected into the oocytes. Several authors reported that HEPES significantly increases intracellular pH (14, 20, 21), which is associated with the inhibition of meiosis reinitiation (17). The alteration of meiosis phases may determine damage in the spindle functions, with abnormal chromosome segregation during the second meiotic division after fertilization, and in the first embryo mitotic division, with the increase of embryos with chromosomal aberration. The significant increase of triploid embryos when a medium with HEPES was used with ICSI is intriguing. Triploid 4

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embryos in ICSI are mostly due to a nondisjunction and extrusion of the second polar body. This may be due in our case to an altered function of the cytoskeleton and meiotic spindle after oocyte activation and fertilization. We can speculate that the injection of HEPES inside the oocyte may modify its function, increasing cellular pH and in some cases enhancing triploid embryo. A well trained embryologist proficient in microinjection can perform ICSI in a few (3–5) minutes, therefore avoiding a long standing time of the gametes under the microscope outside the incubator. This permits the use of a medium buffered with bicarbonate, without HEPES, for all the steps of ICSI, from fertilization throughout the embryo culture, without the stress of changing the medium. In some cases after microinjection we measured the pH of the medium used for the micromanipulation, and we did not observe significant differences between the HEPES-buffered medium and the bicarbonate only– buffered medium. Furthermore, the policy in our center is to microinject no more than ten eggs per patient, and in this study we excluded patients undergoing testicular sperm extraction, because in these cases finding sperm in the testicular tissue may require that eggs stay for a longer time under the microscope. In these cases we still use a HEPESbuffered medium, even though for all other cases we do not routinely use this medium. Even though more studies are needed to elucidate the effects of the HEPES buffer on embryo development, our data suggest that the use of HEPES-buffered media should be reconsidered, especially in established IVF programs with Vol. xx, No. x, Month 2006

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experienced embryologists, in order to obtain a better embryo development after ICSI procedure. REFERENCES 1. Gardner DK, Vella P, Lane M, Wagley L, Schlenker T, Schoolcraft WB. Culture and transfer of human blastocysts increases implantation rates and reduces the need for multiple embryo transfers. Fertil Steril 1998;69:84 – 8. 2. Gardner DK. Development of serum-free media for the culture and transfer of human blastocysts. Hum Reprod 1998;13(Suppl 4):218 –25. 3. Van Langendonckt A, Demylle D, Wyns C, Nisolle M, Donnez J. Comparison of G1.2/G2.2 and Sydney IVF cleavage/blastocyst sequential media for the culture of human embryos: a prospective, randomized, comparative study. Fertil Steril 2001;76:1023–31. 4. Cooke S, Quinn P, Kime L, Ayres C, Tyler JR, Driscoll GL. Improvment in early human embryo development using new formulation sequential stage-specific culture media. Fertil Steril 2002;78:1254 – 60. 5. Iwasaki T, Kimura E, Totsukawa K. Studies on a chemically defined medium for in vitro culture of in vitro matured and fertilized porcine oocytes. Theriogenology 1999;51:709 –20. 6. Escriba MJ, Silvestre MA, Saeed AM, Garcia-Ximenez F. Comparison of the effect of two different handling media on rabbit zygote developmental ability. Reprod Nutr Dev 2001;41:181– 6. 7. Li M, Farley RA, Lester HA. Voltage-dependent transient currents of human and rat 5-HT transporters (SERT) are blocked by HEPES and ion channel ligands. FEBS Lett 2002;513:247–52. 8. Lee MA, Storey BT. Bicarbonate is essential for fertilization of mouse eggs: mouse sperm require it to undergo the acrosome reaction. Biol Reprod 1986;34:349 –56. 9. Bhattacharyya A, Yanagimachi R. Synthetic organic pH buffers can support fertilization of guinea pig eggs, but not as efficiently as bicarbonate buffer. Gamete Res 1988;19:123–9. 10. Ali J, Whitten WK, Shelton JN. Effect of culture systems on mouse early embryo development. Hum Reprod 1993;8:1110 – 4.

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11. Suzuki K, Ebihara M, Nagai T, Clarke NG, Harrison RA. Importance of 62 bicarbonate/CO2 for fertilization of pig oocytes in vitro, and synergism 63 with caffeine. Reprod Fertil Dev 1994;6:221–7. 64 12. Pollard JW, Plante C, Leibo SP. Comparison of development of pig zygotes and embryos in simple and complex culture media. J Reprod 65 Fertil 1995;103:331–7. 66 13. Downs SM, Mastropolo AM. Culture conditions affect meiotic regulation in cumulus cell– enclosed mouse oocytes. Mol Reprod Dev 1997; 67 46:551– 66. 68 14. Hamaguchi MS, Watanabe K, Hamaguchi Y. Regulation of intracellu69 lar pH in sea urchin eggs by medium containing both weak acid and base. Cell Struct Funct 1997;22:387–98. 70 15. Quinn P, Warnes GM, Kerin JF, Kirby C. Culture factors in relation to 71 the success of human in vitro fertilization and embryo transfer. Fertil Steril 1984;41:202–9. 72 16. Palermo G, Joris H, Devroey P, et al. Pregnancies after intracytoplas- AQ: 3 73 mic injection of single spermatozoon into an oocyte. Lancet 1992;340: 74 17– 8. 17. Picard A, Doree M. Intracellular microinjection of alkaline buffers 75 reversibly inhibits the initial phase of hormone action in meiosis reini76 tiation of starfish oocytes. Dev Biol 1983;97:184 –90. 18. Veeck LL. An atlas of human gametes and conceptus. London (UK): 77 Parthenon Publishing; 1999. 78 19. Keskintepe L, Brackett BG. In vitro developmental competence of in 79 vitro–matured bovine oocytes fertilized and cultured in completely defined media. Biol Reprod 1996;55:333–9. 80 20. Zigler JS Jr, Lepe-Zuniga JL, Vistica B, Gery I. Analysis of the 81 cytotoxic effects of light-exposed HEPES-containing culture medium. In Vitro Cell Dev Biol 1985;21:282–7. 82 21. Bowman CM, Berger EM, Butler EN, Toth KM, Repine JE. HEPES 83 may stimulate cultured endothelial cells to make growth-retarding ox84 ygen metabolites. In Vitro Cell Dev Biol 1985;21:140 –2. 22. Mahadevan MM, Fleetham J, Church RB, Taylor PJ. Growth of mouse 85 embryos in bicarbonate media buffered by carbon dioxide, HEPES, or 86 phosphate. J In Vitro Fert Embryo Transf 1986;3:304 – 8.

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Use of a medium buffered with n-hydroxyethylpiperazine-n-ethanesulfonate (HEPES) in intracytoplasmic sperm injection procedures is detrimental to the outcome of in vitro fertlization F. Morgia, M. Torti, M. Montigiani, C. Piscitelli, A. Giallonardo, M. Schimberni, P. Giannini, and M. Sbracia Rome, Italy The use of a medium buffered with Nhydroxyethylpiperazine-N-ethanesulfonate for microinjection of sperm into oocytes is detrimental to the outcome of in vitro fertilization when compared with a medium buffered only with bicarbonate.

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