Ovarian stimulation, in vitro fertilization, and effects of culture conditions on baboon preimplantation embryo development

Ovarian stimulation, in vitro fertilization, and effects of culture conditions on baboon preimplantation embryo development Tien-cheng Chang, Ph.D.,a Carlton A. Eddy, Ph.D.,a Ying Ying, Ph.D.,b Ya-guang Liu, M.D.,c Alan E. Holden, Ph.D.,d Robert G. Brzyski, M.D., Ph.D.,a and Robert S. Schenken, M.D.a a

Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, University of Texas Health Science Center, San Antonio, Texas; b Department of Obstetrics and Gynecology, University of South Florida, Tampa, Florida; c Division of Reproductive Research, Department of Obstetrics and Gynecology, University of Texas Health Science Center, San Antonio, Texas; and d Section of Social Sciences, Department of Obstetrics and Gynecology, University of Texas Health Science Center, San Antonio, Texas

Objective: To evaluate the effects of ovarian stimulation and intracytoplasmic sperm injection (ICSI)-induced fertilization and efficacy of various culture systems on in vitro development of baboon embryos. Design: In vitro study, animal model. Setting: Research laboratory. Animal(s): Baboons in laboratory animal research facility. Intervention(s): Baboons received FSH (75 IU daily) for 7 to 8 days and FSH/LH (75/75 IU daily) for 3 days, followed by hCG (2,000 IU). Oocytes were retrieved laparoscopically 36 hours after hCG. Intracytoplasmic sperm injection was performed on metaphase II (MII) oocytes. Fertilized embryos were placed into different culture conditions and feeder cell coculture. Embryo development was observed through the most advanced stages, including blastocyst formation. Main Outcome Measure(s): Oocytes retrieved, fertilization rates, multicell embryo rates, and blastocyst rates. Result(s): Baboon oocytes (n ¼ 1,924, from 49 cycles) were retrieved. Significant heterogeneity was seen in ovarian response to exogenous gonadotropins and subsequent oocyte maturation. The percentage of MII oocytes showed no significant difference among individual female baboons and stimulation cycles. Nearly two thirds of MII oocytes were successfully fertilized with ICSI. Blastocyst rates varied significantly among embryos in different treatments. Coculture with feeder cells in P1/Blast, Quinn’s Advantage, and Sydney IVF media generated better blastocyst rates. Conclusion(s): We tested multiple media and feeder cell combinations to optimize culture conditions in baboon embryo culture and obtained a high blastocyst rate similar to those reported for rhesus monkey embryos cultured in vitro, but still lower than with assisted reproductive technologies in women. (Fertil Steril 2011;95:1217–23. 2011 by American Society for Reproductive Medicine.) Key Words: Nonhuman primate, baboon, assisted reproductive technology, ovarian stimulation, IVF, ICSI, embryo culture, blastocyst

Nonhuman primates (NHP) have been widely used for studying human reproductive physiology and assisted reproduction. Considerable effort has been given to the development of follicle stimulation protocols and optimization of sperm capacitation and fertilization events, leading to successful NHP IVF in the early 1980s (1, 2). To date, hundreds of NHPs have been born by assisted reproductive technologies (ARTs) worldwide (3). There are more than 200 species of NHPs, but only a limited number have been used for research in reproductive biology. Most Received April 16, 2010; revised June 25, 2010; accepted June 29, 2010; published online August 11, 2010. T.-c.C. has nothing to disclose. C.A.E. has nothing to disclose. Y.Y. has nothing to disclose. Y.-g.L. has nothing to disclose. A.E.H. has nothing to disclose. R.G.B. has nothing to disclose. R.S.S. has nothing to disclose. Supported by the AT&T Foundation. Presented at the 42nd Annual Meeting of the Society for the Study of Reproduction, July 18–22, 2009, Pittsburgh, Pennsylvania. Reprint requests: Tien-cheng Chang, Ph.D., Department of Obstetrics and Gynecology, University of Texas Health Science Center, 7703 Floyd Curl Drive, MC7836, San Antonio, TX 78229 (E-mail: [email protected]).

0015-0282/$36.00 doi:10.1016/j.fertnstert.2010.06.095

studies used Old World monkeys, including the rhesus macaque, cynomolgus macaque, squirrel monkey, baboon, and a New World primate, the common marmoset. Assisted reproductive technologies in Old World NHPs provide a unique opportunity for studying fertilization mechanisms, early embryo development, embryonic stem cell derivation for regenerative medicine, and the creation of clones by embryo splitting and nuclear transplantation. Furthermore, the combination of ART and transgenic technology will promote the development of transgenic NHP for studying human inherited genetic disorders (4–8) and mitochondrial gene replacement (9), as well as modeling implantation and the interaction between the embryo with extracellular matrix (10–13) and the maternal endometrial environment (14–18). The baboon (Papio cynocephalus) is emerging as an important and relevant experimental model with which to examine a wide range of pre- and postimplantation biological processes. However, ART in baboons is not as well established as with other Old World NHP models, such as rhesus and cynomolgus monkeys. Applying human and rhesus monkey ART protocols to baboons has not provided the same blastocyst development rate expected (19).

Fertility and Sterility Vol. 95, No. 4, March 15, 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.

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The purpose of this study was to evaluate the efficacy of different IVF and in vitro culture techniques on preimplantation baboon embryo development and for modeling human reproductive biology and regenerative medicine. We investigated the effects of ovarian stimulation, intracytoplasmic sperm injection (ICSI)-induced fertilization, and various culture systems on in vitro development of the baboon embryo through the blastocyst stage.

FIGURE 1 Ovarian stimulation response of female baboon in different cycles. Repeated ovarian stimulation (OS) cycles resulted in (A, B) fewer retrieved oocytes and MII oocytes, but (C) similar percentages of MII oocytes. N ¼ number of baboons; box plot indicates mean  1 SD and range of minimum and maximum values.

MATERIALS AND METHODS Ovarian Stimulation and Follicle Retrieval from Baboon Ovaries Individually caged adult healthy fertile female baboons at day 1 or 2 after menses received recombinant (r) human gonadotropin (rFSH, 75 IU/d; Serono, Geneva, Switzerland) for 7 to 8 days and rFSH þ rLH (75 IU/d each; Serono) for 3 days, followed by a single injection of hCG (2,500 IU; Serono). All animal protocols conducted in this experiment were approved by the Institutional Animal Care and Use Committee, University of Texas Health Science Center at San Antonio, San Antonio, Texas.

Oocyte Collection and Preinsemination Culture Laparoscopic surgery and follicular aspiration followed 35–37 hours after hCG injection. Follicular fluid aspirates were examined under a stereomicroscope (Olympus, Tokyo, Japan) to identify cumulus–oocyte complex. Cumulus cells were removed using embryo concentrator filter (Em Con filter, Immuno Systems, Spring Valley, WI) with addition of hyaluronidase (Sigma, St. Louis, MO). Oocyte morphology, maturation stage, and the presence of first polar body of cumulus cell–free oocytes were assessed. Oocytes were moved into a four-well plate (Nunc, Roskilde, Denmark) of buffalo rat liver (BRL) feeder cells in P1 medium (Irvine Scientific, Santa Ana, CA) supplemented with 5% defined fetal bovine serum (FBS; HyClone, Logan, UT) and placed in an incubator with 37 C triple gas (5% CO2, 5%O2, 90%N2).

Sperm Collection and Processing Male baboons were sedated with ketamine. Semen specimens were collected by rectal probe electroejaculation and processed in sperm washing medium on the day of oocyte retrieval. Specimens were allowed to liquefy at room temperature for 30 minutes. The liquid portion was aspirated from the coagulum and transferred to a sterile conical tube. Semen specimen was processed by swimup for 45–60 minutes, then washed twice in sperm-washing medium (modified human tubal fluid [mHTF] N-2-hydroxyethylpiperazine-N0 -2-ethanesulfonic acid [HEPES]-buffered medium supplemented with 5 mg/mL human serum albumin [Irvine Scientific]) at 200–300  g centrifugation.

BRL Feeder Cell Culture and Preparation Buffalo rat liver cells were used in coculture with oocytes, zygotes, and preimplantation embryos. Frozen BRL cells (American Type Culture Collection, Manassas, VA) were thawed and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA) supplemented with 10% FBS in T25 tissue culture flasks (Corning, Lowell, MA) and incubated at 37 C with 5% CO2. Subculture of BRL cells was performed weekly by trypsinizing BRL cells using 0.05% trypsin (Invitrogen). To prepare monolayer BRL for embryo culture, cells were plated at a density of 1  104 per well in Nunc four-well plates in DMEM and incubated for 24–36 hours before oocyte retrieval. Eight to twelve hours before oocyte retrieval, DMEM was replaced with oocyte/embryo culture media and covered by light paraffin oil (Sage IVF, Cooper Surgical, Trumbull, CT).

IVF by ICSI Intracytoplasmic sperm injection was performed on metaphase II (MII)-stage oocytes using an inverted microscope (Nikon, Tokyo, Japan) with Hoffman modulation contrast, heating plate (Tokai Hit, Fujinomiya, Japan), micromanipulators (Narishige, Tokyo, Japan), and microinjectors connected to a holding pipette (95–120-mm outer diamter, 15–20-mm inner diameter; Humagen, Charlottesville, VA) and a microinjection pipette (7–8-mm outer diameter,

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5–6-mm inner diameter, with 30 11–12-mm-length beveled tip; Humagen). Each oocyte was placed in mHTF HEPES-buffered medium covered with paraffin oil. A single baboon sperm in 15% polyvinylpyrrolidone (Irvine Scientific) with normal morphology was selected and injected into oocyte.

Embryo Culture After ICSI, oocytes were rinsed in mHTF HEPES-buffered medium and placed in different culture media, including G series (Vitrolife, Goteborg, Sweden), Global (LifeGlobal, Guilford, CT), P1 (Irvine Scientific), Quinn’s

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FIGURE 2 Development of ICSI-derived baboon preimplantation embryos. (A) Fertilized baboon oocytes with two pronuclei identified; (B, C) cleavagestage embryos; (D) blastocyst with inner cell mass and trophectoderm. Scale bars ¼ 100 mm.

Chang. Baboon OS, IVF, and embryo development. Fertil Steril 2011.

Advantage (Sage IVF, Cooper Surgical), and Sydney IVF (Cook Medical, Bloomington, IN), as well as rhesus monkey preimplantation embryo culture media CMRL-1066 and HECM-9, supplemented with 10% FBS or serum replacement solution and amino acids, and covered by light paraffin oil. A monolayer of BRL feeder cells in combination with test media was used in some of the treatment groups to examine the effect of coculture. The plates were returned to incubator at 37 C with triple gas.

analyzed using analysis of variance. Next, the effect of different media with and without feeder cells on the percentages of fertilization, multicellstage embryos per fertilized oocytes, and blastocysts per multicell-stage embryos were compared using analysis of variance with Sidak’s adjustment for multiple comparisons. Results are reported as mean  SD.

RESULTS Confirmation of Fertilization by Pronuclei Examination Two pronuclei and protrusion of the second polar body were identified by 16–18 hours after ICSI. Zygotes with normal fertilization were selected and returned to culture.

Extended Culture of Eight-Cell- Through Blastocyst-Stage Baboon Embryos On day 3 after ICSI, embryos cultured in sequential media, such as G series, Blast (P1/Blast; Irvine Scientific), Quinn’s Advantage, and Sydney IVF, were transferred to new four-well plates containing blastocyst media from each manufacturer supplemented with BRL feeder cells if applicable. Medium plates were changed every 2 days through blastocyst formation. Embryos cultured in Global medium, CMRL-1066, and HECM-9 were refreshed every 2 days in new medium plates for continuous culture. Embryo development was observed daily through the most advanced stages, including blastocyst formation, or until development stalled.

Data Analysis Mean number of retrieved oocytes, number of retrieved mature MII oocytes, and percentage of mature MII oocytes for numbers of stimulation were

Fertility and Sterility

We obtained 1,924 baboon oocytes from 49 cycles of ovarian stimulation. Significant heterogeneity was seen in ovarian response to exogenous gonadotropins and oocyte maturation among individual baboons receiving gonadotropin stimulation (retrieved oocytes, 39.3  27.0; range, 3–129). The number of mature MII stage oocytes retrieved from ovarian stimulation also varied significantly among baboons (18.4  14.8; range, 0–59). Baboons going through the second and third cycles of ovarian stimulation had poorer follicle development and lower numbers of MII oocytes as compared with their first cycles of ovarian stimulation, despite receiving the same dosages of gonadotropin injections. There was a significant difference between the first (OS#1) and second (OS#2) cycles in both retrieved oocyte numbers (P<.05) and MII oocyte numbers (P<.05) (Fig. 1A and B). Only two animals had a third stimulation (OS#3), and no statistical analyses were performed in this subset. Despite the significant difference in retrieved and MII oocyte numbers between repeated ovarian stimulation cycles, the percentages of MII-stage oocytes from each baboon through repeated cycles of stimulation were not significantly different. In addition, the percentage of MII oocytes retrieved showed no significant difference

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FIGURE 3 Effect of medium and coculture combinations on (A) fertilization rates, (B) cleavage rates, and (C) comparative blastocyst rates per cleavage-stage multicell embryos (mean percentages  SD). *The blastocyst rates were significantly better with Sydney IVF medium supplemented with BRL feeder cells and P1/Blast medium with BRL feeder cells.

rates of multicell-stage embryos from fertilized zygotes showed some variability among baboons but no significant difference among culture conditions with or without BRL feeder cells (Fig. 3B). Many embryos stalled at the morula stage, resulting in low blastocyst formation rates (blastocyst per multicell embryo, 12.4%, 56 of 404; 13.1% with BRL feeder cells and 11.8% without BRL feeder cells) (Fig. 3C). All culture systems resulted in comparable rates of development through fertilization and morula formation (Fig. 3A and B); the blastocyst formation rates ranged from none to 37.7%. In cultures without feeder cells, the blastocyst rate was higher with Sydney IVF as compared with all other media (Fig. 3C). Coculture with feeder cells in P1/Blast, Quinn’s Advantage, and Sydney IVF media generated better blastocyst rates, with Sydney IVF being better than all other media (P<.05) (Fig. 3C). The blastocyst rate for embryos cultured in P1/Blast media with BRL cells monolayer (30%  27.4%, 7 of 37) was significantly better than that observed without BRL cells (4.2%  11.8%, 4 of 47) (P<.05). Sydney IVF medium with BRL feeder cell coculture also provided a slightly higher blastocyst rate (37.7%  9.7%, 13 of 36) than Sydney IVF medium alone (33.0%  0, 5 of 21), but the difference was not significant. Coculture with BRL feeder did not significantly increase blastocyst rates with any other media. In addition, the morphological quality of blastocysts in coculture was also better when compared with embryos without coculture. Blastocyst rates were lower with Global and Quinn’s Advantage media (6.7%  16.3%, 2 of 22, and 16.1%  20.1%, 16 of 91, respectively) and even lower with coculture (0 of 30 and 11.1%  17.2%, 2 of 32, respectively). Embryos cultured in HECM9 medium had lower fertilization rates (45.4%  26.7%, 50 of 148) than other groups (overall 63.3%  22.6%, 496 of 844) but a blastocyst rate from cleavage-stage embryos (13.3%  25.7%, 6 of 26) comparable to that of other groups (overall 12.4%  19.5%, 56 of 404). The blastocyst rates were significantly better (P<.05) with Sydney IVF medium supplemented with BRL feeder cells (37.7%  9.7%, 13 of 36) and P1/Blast medium with BRL feeder cells (30.0%  27.4%, 7 of 37) (Fig. 3C, see asterisks).

DISCUSSION

Chang. Baboon OS, IVF, and embryo development. Fertil Steril 2011.

among individual female baboons (46.6%  25.8%, 900 of 1,924) (Fig. 1C). Nearly two thirds of MII oocytes were successfully fertilized with ICSI (63.5%  22.7%, 496 of 844), and the majority developed to the multicell stage (87.0%  28.1%, 446 of 488) (Fig. 2). Oocytes injected with sperm and cultured in different media with or without BRL feeder cells showed similar fertilization rates (Fig. 3A). The

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Assisted reproductive technologies have been applied in several NHP species, including rhesus monkeys (2, 5–7, 13, 20–70), cynomolgus monkeys (71–78), African green monkeys (vervet) (71, 79, 80), squirrel monkeys (81–88), marmosets (89–93), great apes (94), and baboons (1, 19, 84, 95–102). Various levels of success in these species indicated the fundamental differences in gametogenesis, preimplantation embryo development, and nutrition requirement for optimal in vitro embryo development (103, 104). Directly applying human ART protocols to NHPs does not produce the same results seen in humans, and modifications are needed to enhance embryo development in NHPs. These may include stimulation regimens for follicular development, sperm activation and subsequent fertilization events, oocyte maturation, and media requirements for preimplantation embryo development. Recombinant hormones commonly used in human ART may be antigenic in baboons as well as other macaque species, and consequently repeated stimulation protocols often result in poor responses and a low number of oocytes (105, 106). Macaquespecific FSH and CG are not commercially available at this time. Future development may be necessary to optimize macaque ovarian stimulation protocols. Assisted reproductive technology protocols in baboon, including ovarian stimulation, IVF, and specifically embryo culture to reach

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blastocyst stage, have been less successful than observed with rhesus monkeys (19, 95, 96, 98, 107). There seems to be a block at the morula stage, where the rate of postcompaction embryo showing differentiation into inner cell mass and trophectoderm cell lineages decreases dramatically. Here we assessed the effects of several embryo culture media with and without feeder cell monolayer supplement on baboon embryo development. The combination of BRL cell coculture with Sydney IVF medium and P1/Blast media consistently promoted the highest percentages of baboon preimplantation embryos to reach blastocyst stage. These results are higher than in previous baboon ART studies (19, 96, 108) and comparable to more established NHP ART protocols used in the rhesus monkey (3, 106). The rates of mature oocytes retrieved in our study (46.6%  25.8%) were similar to those in a previous study in baboons (17%–63%) (19). Heterogeneous responses to the ovarian stimulation protocol in individual female baboons were also observed in previous studies in baboon and rhesus monkeys (19, 53, 70). The sex skin swelling pattern in the second and third ovarian stimulation cycles of the baboon consistently shows deturgescence, indicating poor stimulation. Interestingly, the percentage of oocyte maturation in baboons receiving more than one ovarian stimulation cycle remained consistent throughout repeated stimulation cycles in this study, which may involve an unknown regulatory mechanism unaffected by immune response to human recombinant gonadotropin administration. Most culture conditions in our treatment groups yielded good fertilization, with two thirds of mature oocytes fertilized (63.3%) compared with previously reported data (range 23-54%, and 39%) (19, 101). Among the media and feeder cell coculture combination tested, the highest fertilization groups were in Global medium with BRL feeder cell (76.4%), Sydney IVF medium with BRL (70.0%), and CMRL medium with BRL (68.6%), but the difference was not statistically significant. Coculture with feeder cells (e.g., BRL, green monkey kidney) were used for rhesus monkey embryo culture in vitro (44, 59, 95). In our study, the BRL feeder cell effect varied dramatically with

each individual medium. Embryos cultured in P1/Blast and Sydney IVF media combining with BRL cells yielded a higher percentage of blastocysts than those cultured in the same media without feeder cells, whereas embryos cultured in other types of media (Global and Quinn’s Advantage) did not show higher blastocyst rates, and in some cases showed lower rates, when feeder cell coculture was present. Feeder cells were always used in the CMRL protocol, and the baboon embryo development was among the lowest. No feeder cells were used with HECM9 medium, and baboon embryos and blastocyst rates were comparable to those seen with Quinn’s Advantage without feeder cells but lower than rates with P1/Blast supplemented with feeder cells and both groups of Sydney IVF treatment. Detailed information of components in each clinical media is proprietary. Consequently, it is impossible to assess which individual components in the media may be responsible for these observations. Our results suggested a unique nutrient requirement by baboon embryos, and further investigation into modifying components in the culture medium (e.g., essential amino acids) may improve the yield of ICSI-derived baboon blastocysts. This also provides an opportunity to understand how various components in the media may be critical for NHP embryo development. In conclusion, Sydney IVF and P1/Blast media with BRL feeder cells resulted in higher blastocyst rates than all other media/feeder cell treatments we tested. The blastocyst rates were similar to those reported for rhesus monkey embryos cultured in vitro (3, 106) but slightly lower than those with clinical ART in women (109, 110). We are investigating other ovarian stimulation protocols and in vitro embryo culture systems to enhance the success rates of embryo development for future application of this unique experimental model to study human reproductive biology and regenerative medicine. Acknowledgment: The authors thank Peter Binkley and the Department of Laboratory Animal Research of the University of Texas Health Science Center at San Antonio for technical and surgical procedure support.

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