Hyaluronic acid can successfully replace albumin as the sole macromolecule in a human embryo transfer medium

Transcription

1 FERTILITY AND STERILITY VOL. 79, NO. 6, JUNE 2003 Copyright 2003 American Society for Reproductive Medicine Published by Elsevier Inc. Printed on acid-free paper in U.S.A. Hyaluronic acid can successfully replace albumin as the sole macromolecule in a human embryo transfer medium Alex Simon, M.D., Anat Safran, Ph.D., Ariel Revel, M.D., Einat Aizenman, Ph.D., Beni Reubinoff, M.D., Anat Porat-Katz, B.Sc., Aby Lewin, M.D., and Neri Laufer, M.D. IVF Unit, Department of Obstetrics and Gynecology, Hadassah University Hospital Ein Kerem, Jerusalem, Israel Objective: To examine the effect on pregnancy and implantation rates when highly purified, fermentationbased hyaluronic acid was the only macromolecule supplement to the transfer medium in a human IVF program. Design: Prospective randomized study. Setting: In vitro fertilization center in an academic medical institution. Patient(s): Eighty patients were included in this prospective randomized double blind study. Inclusion criteria were age 35 years, the availability of at least three embryos eligible for transfer on day 3 after fertilization, and no more than three previous embryo transfer attempts. Intervention(s): All embryos were cultured in P1 medium containing 10% synthetic serum substitute (SSS) until day 3. Patients were randomly allocated to two groups; in treatment group A (40 patients), embryos were transferred to P1 medium supplemented with 0.5 mg/ml hyaluronic acid for 5 10 min before their intrauterine transfer. In the control group B (40 patients), embryos were transferred, as routinely performed, in P1 medium containing 10% SSS. Main Outcome Measure(s): Clinical pregnancy and implantation rates. Result(s): The mean age of the female partner was years and years for groups A and B, respectively. In group A, 103 embryos were transferred and in group B, 97 embryos were transferred for a similar mean number of and embryos/transfer, respectively. Twenty-five pregnancies were achieved in group A, and 21 pregnancies in group B. This led to a comparable clinical pregnancy and implantation rates of 62.5% and 34% as compared to 52% and 26.8% for groups A and B, respectively. Conclusion(s): Hyaluronic acid can successfully replace albumin as a sole macromolecule in a human embryo transfer medium and result in high pregnancy and implantation rates. The use of this supplement is an important step in the development of human embryo culture media free of blood-derived additives. (Fertil Steril 2003;79: by American Society for Reproductive Medicine.) Key Words: Culture medium, embryo transfer, hyaluronic acid, hyaluronan, IVF Received July 15, 2002; revised and accepted October 24, Reprint requests: Alex Simon, M.D., Department of Obstetrics and Gynecology, Hadassah University Hospital, Ein Kerem, P.O. Box 12000, Jerusalem 91120, Israel (FAX: ; asimon@md.huji. ac.il) /03/$30.00 doi: /s (03) In vitro fertilization programs use commercially prepared media for oocyte retrieval, embryo culture, sperm preparation, and embryo transfer. From the early beginnings of IVF it has been recognized that culture media should be supplemented with proteins to optimize the culture conditions. The physiological functions of plasma proteins are well documented and include a direct role in osmoregulation. Proteins also serve as a source of energy and as reservoirs for the release of hormones, vitamins, and metals. Because albumin is highly abundant in the female reproductive tract (1), it has traditionally served as the main macromolecule in most culture media used for in vitro growth of human embryos. In addition, albumin confers on the culture medium the useful physical properties of lubrication and viscosity, promoting ease of handling the embryo and preventing its adherence to the culture dish. The sources of the proteins, especially albumin, for culture media are the patient s own serum, fetal cord serum, commercially pooled human serum albumin (HSA), and lately, recombinant HSA. The use of blood-derived albumin, however, raises concern about both biological variation and the possibility of disease transmission through vi- 1434

2 ral and prion vectors (2, 3). Therefore, the search for other macromolecules that will successfully substitute for bloodderived albumin in culture media without compromising embryo growth and development is to be encouraged. Several macromolecules such as polyvinylpyrolidone, polyvinylalcohol, and hyaluronic acid (hyaluronan) have been suggested as alternatives. Although the potential of polyvinylpyrolidone and polyvinylalcohol macromolecules to efficiently promote embryo development in vitro is still questionable (4, 5), the efficacy of hyaluronic acid in supporting mouse embryo development and growth is well established (6, 7). In addition, the teratological properties of both polyvinylpyrolidone and polyvinylalcohol, which have not been fully examined, raise concerns regarding their use in humanassisted reproduction (8). In contrast, hyaluronan is a naturally existing macromolecule related to the glycosaminoglycans family that is abundant in human fluid secretions and extracellular matrix (9). Hyaluronan is a linear polysaccharide of alternating D-glucoronic acid N-acetyl-D-glucosamine residues and exists physiologically in the reproductive system in cervical mucus, the cumulus, follicular fluid (FF), and seminal plasma (10 12). Interestingly, both human (13) and bovine (14) embryos possess a surface receptor to hyaluronan that can be detected throughout their development up to the blastocyst stage. Furthermore, it has been shown that hyaluronan increases in concentration at the time of implantation of mouse embryos (15). In addition, hyaluronan, when supplemented to sperm preparation media, increases sperm motility and improves retention of sperm motility in long-term incubation of both fresh and cryopreserved/thawed human spermatozoa (16). These findings support the assumption that hyaluronan may play a physiological role in sperm motility, early embryo development, and implantation. Thus, hyaluronan may be considered as an ideal macromolecule to replace albumin in culture media. Recent studies have demonstrated that mouse embryos can successfully grow and implant when cultured and transferred in media containing hyaluronan extracted from rooster comb (6) or as a recombinant substance (7). In a previous study, using a fermentation-based hyaluronic acid from a bacterial source in an animal mouse model, we found that the inclusion of hyaluronan as the only macromolecule in the transfer medium did not hamper the implantation or fetal development rates when compared to controls (Simon et al., unpublished data). The aim of this study was to evaluate the effect of highly purified, fermentation-based hyaluronic acid on the implantation of human embryos when it is used as the sole macromolecule in the transfer medium. MATERIALS AND METHODS Ovarian Stimulation Ovarian stimulation was carried out for all patients by a desensitizing protocol using GnRH-agonist (GnRH-a) D-Trp-6-LHRH (Decapeptyl, Lapidot Pharmaceuticals Ltd, Herzelia, Israel). Patients were treated by Decapeptyl 0.1 mg/day given SC for 14 days from the midluteal phase of the preceding cycle (days 21 23) to suppress the pituitary function. As all patients were ovulatory, they experienced menses with the Decapeptyl treatment. After pituitary downregulation was confirmed by 17- E 2 serum levels 100 pmol/l (converting factor 3.67 for pg/ml), serum P 2 nmol/l (converting factor 3.18 for ng/ml), and the absence of any ovarian follicle of 10 mm in size, patients underwent gonadotropin stimulation. Induction of ovulation with recombinant FSH (Gonal-F 75, Serono, Geneva, Switzerland) was started, given SC in a dosage of two ampules per day (150 U of recombinant FSH) for the first 5 days. After the fifth day of ovarian stimulation, the dose of recombinant FSH was tailored according to the individual daily response based on transvaginal ultrasound, E 2, and P monitoring. When serum E 2 level was 2,000 pmol/l and at least two follicles were 17 mm in diameter, 5,000 10,000 U of hcg (Chorigon, Serono) were administered IM. Transvaginal oocyte retrieval was performed hours after hcg administration under ultrasound guidance. The retrieved oocytes were then fertilized according to sperm quality by either conventional IVF or intracytoplasmic sperm injection (ICSI) as previously described (17). Fertilized oocytes were cultured in P1 medium (Irvine Scientific, Santa Ana, CA) enriched with 10% synthetic serum substitute (SSS, Irvine Scientific) until day 3 of fertilization when embryo transfer took place. Patient Selection and Embryo Transfer Eighty patients were recruited on the day of their embryo transfer to sign an informed consent and be included in this prospective study that had been previously approved by the local ethics committee. To be eligible for inclusion, a patient had to be 35 years old or younger, had to have at least three embryos suitable for transfer, and had had no more than three previous embryo transfer failure attempts. After informed consent was given, patients were randomly divided into two groups. In group A (40 patients), embryos were transferred to P1 medium supplemented with 0.5 mg/ml of highly purified fermentation-based hyaluronic acid (Biolon, Bio-Technology General Ltd., Kiryat Weizman, Rehovot, Israel) for 5 10 minutes before intrauterine transfer took place. In group B that served as control (40 patients), embryos were transferred, as routinely applied, in P1 medium containing 10% SSS. All embryo transfers were performed using a Casmed catheter (Casmed International Ltd., Surrey, UK) while the physician was blinded to the transfer medium. FERTILITY & STERILITY 1435

3 TABLE 1 Clinical outcome of embryo transfers in study groups A and B. Transfer in HA (group A) Transfer in SSS (group B) P value Patient s age Transfers Embryos transferred Embryos/transfer Clinical pregnancies Singleton Twin (%) 10 (40) 5 (23.8) Ongoing pregnancies Clinical pregnancy rate per ET 62.5% 52.0% Implantation rate 34.0% 26.8% Ongoing pregnancy rate per ET 57.5% 47.5% Deliveries Newborns Note: Values are mean SD. HA hyaluronic acid; SSS synthetic serum substitute. P value is not significant. Simon. Hyaluronan for embryo transfer. Fertil Steril The luteal phase was supported by intravaginal micronized P (Uterogestan, Besins-Iscovesco, Paris, France) in three divided doses for a total amount of 900 mg/day starting 1 day after oocyte retrieval. Patients were tested for serum -hcg assay 14 days after embryo transfer. If the pregnancy test was positive, patients were followed with serial ultrasounds to determine fetal viability. Clinical pregnancy was defined as the presence of a gestational sac on transvaginal ultrasound. When pregnancy occurred, luteal support was continued until 10 weeks gestation. Statistical Analysis Clinical results of the embryo transfer cycles were compared between the groups. Data are presented as mean standard deviation (SD). The results were analyzed using the 2 test or Student s t-test as appropriate and P value of.05 was considered as statistically significant. Statistical analyses were performed using a standard computer program of Microsoft Excel for Windows. RESULTS Eighty patients were included in this prospective randomized double blind study with 40 patients in each study group. The two study groups were similar as to their mean age ( years and years for groups A and B, respectively). Two to three embryos were transferred to each patient resulting in a comparable mean number of embryos transferred (Table 1). The clinical outcome of transfers in each group is indicated in Table 1. In group A, 103 embryos were transferred resulting in 25 clinical pregnancies for a pregnancy rate of 62.5% per embryo transfer and an implantation rate of 34%. These rates were somewhat higher, although not statistically significant, from those obtained in group B, 52% clinical pregnancy rate and 26.8% implantation rate. Although a similar mean number of embryos was transferred in both groups, the multiple gestation rate in group A was higher in comparison to that of group B, 40% to 23.8%. This reflects the somewhat higher implantation rate observed in group A. To date 14 patients of group A and 13 patients of group B gave birth to 21 and 15 healthy newborns, respectively. DISCUSSION This study demonstrates that hyaluronic acid can successfully replace albumin as a sole macromolecule in a human embryo transfer medium resulting in comparably high pregnancy and implantation rates. Traditionally, the source of protein additives in culture media for human IVF, mainly albumin, is from pooled human serum. The use of donor serum proteins, however, raises concerns regarding the possible transmission of pathogenic diseases to patients undergoing assisted reproductive treatments. Indeed, hepatitis B has been reported in patients who received embryos cultured in medium containing pooled sera contaminated with hepatitis B virus (2). More recently, a commercially prepared protein product added to human embryo culture medium was suspected of including blood components from a donor with Creutzfeld-Jacob disease (3). Therefore, the development of culture media that include an efficient macromolecule other than human bloodderived albumin is an important consideration. Hyaluronan is a naturally occurring macromolecule, related to the glycosaminoglycan family that is abundant in tissues including the reproductive system (9, 18). Hyaluronan, as other glycosaminoglycans and proteoglycans, is released into the extracellular matrix and is involved in matrix formation, cell cell and cell matrix adhesion, cell proliferation and migration, and shows co-receptors activity for growth factors (9). When one considers the physical properties and the biologic activity of hyaluronan, it is not surprising that this macromolecule was suggested as a suitable replacement for albumin in human culture media. Several studies have addressed the use of hyaluronic acid in culture media in mammals (6, 7, 19). The effects of albumin and hyaluronic acid extracted from rooster combs on mouse embryo development were analyzed by Gardner et al. (6). Although hyaluronic aacid could be used as the sole macromolecule in the culture medium, an interaction between hyaluronic acid and albumin was evident in promoting embryo development in culture. In a recent study (7), the same investigators have reported on similar rates of blastocysts development, but a higher percentage of inner cell mass (ICM) cells when the culture media contained recombinant HSA (1.25 mg/ml) and re Simon et al. Hyaluronan for embryo transfer Vol. 79, No. 6, June 2003

4 combinant hyaluronic acid (0.125 mg/ml) in comparison to culture media having 5 mg/ml HSA. These results corroborate the finding of Furnus et al. (19) who reported that supplementation of culture media with hyaluronic acid improves the development of in vitro maturation (IVM)/IVF bovine embryos to the blastocyst stage. Assessing the implantation, Gardner et al. (6) reported a significant increase in both implantation and fetal development rates when hyaluronic acid was the only macromolecule in the transfer medium of mouse embryos in comparison to transfer medium that contained no macromolecule at all. In that study, however, the implantation and fetal developmental rate was comparable when the transfer medium was supplemented by either hyaluronic acid and bovine serum albumin or hyaluronic acid alone. Before applying hyaluronic acid for clinical use, we similarly, evaluated the implantation rate and fetal development rate of mouse embryos when the transfer medium contained either hyaluronic acid or albumin. We found that whereas the addition of hyaluronic acid or albumin to transfer media resulted in similar implantation rates, a higher fetal developmental rates were achieved when hyaluronic acid was used in the transfer medium (Simon et al., unpublished data). From these animal model experiments we concluded that the use of hyaluronic acid as the sole macromolecule in the transfer medium has no adverse effect on the implantation rate and might even be found advantageous if applied clinically. In this study, human embryos were cultured for 72 hours in P1 medium enriched with 10% SSS followed by a random transfer in either the same medium or in P1 medium supplemented with 0.5 mg/ml hyaluronic acid. A high and comparable clinical pregnancy rate was achieved in both groups (62.5% and 52% for the hyaluronic acid and SSS groups, respectively). Although the number of embryos transferred was comparable between the groups, the multiple pregnancy rate for hyaluronic acid reached 40%, whereas that of the SSS group was 23%. This reflects on the implantation rate, which was somewhat higher for the hyaluronic acid group as compared to the SSS group (34% and 26.8%, respectively). This difference, however, was not found to be statistically significant. The results of this study indicate that hyaluronic acid can be used as the sole macromolecule in the transfer media and might even be beneficial considering the biological and physical properties of this substance. The use of hyaluronic acid in the transfer medium may offer several advantages in the implantation process. Concern has been expressed about to the immediate or late expulsion of the embryos after their transfer to the uterine cavity (20). Hyaluronan, by its physical property, produces a viscous solution that might enhance the embryo transfer process and prohibit the expulsion of embryos from the uterine cavity after transfer. In addition, it has been suggested that the use of hyaluronic acid in transfer media can facilitate the diffusion and integration of the embryos in the viscous solution characterizing the intrauterine-secreted fluid (6). Moreover, it has been shown that proteoglycans as well as hyaluronic acid are involved in cell cell and cell matrix adhesion, and in activation or inhibition of proteases (9). The role of hyaluronic acid in adhesiveness and embryo implantation is further substantiated by the observation that tumors are highly enriched with hyaluronic acid and that their invasiveness is correlated with hyaluronic acid expression (21, 22). These data and the detection of the hyaluronic acid receptor on mammalian embryos (13, 14) signify the possible role that hyaluronic acid plays in an embryo s attachment, implantation, and development after being transferred to the uterine cavity. We have achieved high pregnancy and implantation rates using either albumin or hyaluronic acid in the transfer media. This study, however, was conducted on a preselected group of patients who were younger than 35 years, had a good ovarian response leading to at least three transferable embryos, and had no history of repeated failures after embryo transfer. The question is whether hyaluronic acid, which has been successfully used in the transfer media of good responder group, would be of advantage for other problematic groups. Clinical studies, as to the value of hyaluronic acid supplementation in transfer media for patients who are poor responders, older than 38 years, and with repeated IVF failure attempts, is currently being evaluated. One might argue that to develop media free of human additives it would be better to culture and transfer the embryos in media supplemented with hyaluronic acid only. However, our experience as well as that of other researchers (6, 19) suggests that although embryos can grow in medium with hyaluronic acid as the sole macromolecule, optimal embryo development is achieved only when both hyaluronic acid and albumin are included in the culture media. Bearing this in mind, we have adopted the practice of culturing the embryos in a standard way and using hyaluronic acid as the sole macromolecule only for embryo transfer. Acknowledging the advantages of hyaluronic acid and the importance of developing culture media lacking blood-derived products, Gardner and Lane (7) have recently reported that recombinant macromolecules (hyaluronan and human albumin) can successfully replace blood-derived HSA for culture media. Clinical studies applying such macromolecule are on the way and we await the results to be published. Our study, along with that performed by Gardner and Lane (7), opens new horizons for the development of culture media free of blood-derived products that can be successfully used for both culture and transfer of human embryos. Note added in proof: Of 25 pregnant patients in group A, 22 gave birth (32 healthy newborns) for a take home baby rate of 55%. This rate was comparable to that of group B in FERTILITY & STERILITY 1437

5 which 18 patients gave birth (22 healthy newborns) for a take home baby rate of 45%. Acknowledgment: The authors thank June Sher for her excellent assistance in revising the manuscript. References 1. Leese HJ. The formation and function of oviduct fluid. J Reprod Fertil 1988;82: Van Os HC, Drogendijk AC. The influence of contamination of culture medium with hepatitis B virus on the outcome of in vitro fertilization pregnancies. Am J Obst Gynecol 1991;165: Kemmann E. Creutzfeldt-Jakob disease (CJD) and assisted reproductive technology (ART). Quantification of risks as part of informed consent. Hum Reprod 1998;13: Cholewa JA, Whitten WK. Development of two-cell mouse embryos in the absence of a fixed-nitrogen source. J Reprod Fertil 1970;22: Biggers JD, Summers MC, McGinnis KL. Polyvinyl alcohol and amino acids as substitutes for bovine serum albumin in culture media for mouse preimplantation embryos. Hum Reprod Update 1997;3: Gardner DK, Rodriegez-Martinez H, Lane M. Fetal development after transfer is increased by replacing protein with the glycosaminoglycan hyaluronan for mouse embryo culture and transfer. Hum Reprod 1999; 14: Gardner DK, Lane M. Recombinant human serum albumin and hyaluronan can replace blood-derived albumin in embryo culture media [abstr]. Fertil Steril 2000;74(Suppl):S Gardner DK, Lane M. Culture of viable human blastocysts in defined sequential serum-free media. Hum Reprod 1998;13(Suppl 3): Salustri A, Camaioni A, Di Giacomo M, Fulop C, Hascall VC. Hyaluronan and proteoglycans in ovarian follicles. Hum Reprod Update 1999;5: Eppig JJ. FSH stimulates hyaluronic acid synthesis by oocyte cumulus cell complexes from mouse preovulatory follicles. Nature 1979;281: Grimek HJ, Bellin ME, Ax RL. Characteristics of proteoglycans isolated from small and large bovine ovarian follicles. Biol Reprod 1984; 30: Binette JP, Ohishi H, Burgi W, Kimura A, Suyemitsu T, Seno N, et al. The content and distribytion of glycosaminoglycans in the ejaculates of normal and vasectomized men. Andrologia 1996;28: Campbell S, Swann HR, Aplin JD, Seif MW, Kimber SJ, Elstein M. CD44 is expressed throughout pre-implantation human embryo development. Hum Reprod 1995;10: Valcarcel A, de Matos DG, Furnus CC. The hyaluronic acid receptor (CD44) is expressed in bovine oocytes and preimplantation stage embryos. Theriogenology 1999;51: Carson DD, Dutt A, Tang JP. Glycoconjugate synthesis during early pregnancy: hyaluronate synthesis and function. Dev Biol 1987;120: Sbracia M, Grasso J, Sayme N, Stronk J, Huszar G. Hyaluronic acid substantially increases the retention of motility in cryopreserved/ thawed human spermatozoa. Hum Reprod 1997;12: Simon A, Revel A, Hurwitz A, Holtzer H, Zentner B-S, Glazer R, et al. Preliminary experience with assisted hatching in advanced- age patients undergoing inracytoplasmic sperm injection. Isr J Obstet Gynecol 1997; 8: Yanagishita M. Proteoglycans and hyaluronan in female reproductive organs. EXS 1994;70: Furnus CC, de Matos DG, Martinez AG. Effect of hyaluronic acid on development of in vitro produced bovine embyos. Theriogenology 1998;49: Mansour RT, Aboulghar MA. Optimizing the embryo transfer technique. Hum Reprod 2002;17: Ropponen K, Tammi M, Parkkinen J, Eskelinen M, Tammi R, Lipponen P, et al. Tumor cell-associated hyaluronan as an unfavorable prognostic factor in colorectal cancer. Cancer Res 1998;58: Anttila MA, Tammi RH, Tammi MI, Syrjanen KJ, Saarikoski SV, Kosma VM. High levels of stromal hyaturonan predict poor disease outcome in epithelial ovarian cancer. Cancer Res 2000;60: Simon et al. Hyaluronan for embryo transfer Vol. 79, No. 6, June 2003