High survival rate of metaphase II human oocytes after first polar body biopsy and vitrification: determining the effect of previtrification conditions

High survival rate of metaphase II human oocytes after first polar body biopsy and vitrification: determining the effect of previtrification conditions Levent Keskintepe, Ph.D.,a,b Yuksel Agca, Ph.D.,c Geoffrey Sher, M.D.,a,b Meral Keskintepe, Ph.D.,d and Ghanima Maassarani, M.D.a a

Sher Institute for Reproductive Medicine, Las Vegas, Nevada, b Department of Obstetrics and Gynecology, School of Medicine, University of Nevada, Reno, Nevada; c Department of Veterinary Pathobiology, University of Missouri–Columbia, Columbia, Missouri; and d ReproCure LLC, Las Vegas, Nevada

Objective: To use metaphase II (MII) bovine oocytes as a model for MII human oocyte cryopreservation and to determine the effect of different previtrification equilibration temperatures, vitrification solutions, zona slitting, and first polar body biopsy on in vitro and in vivo developmental competence of MII human oocytes after the CryoLoop vitrification method. Design: In vitro and in vivo studies. Setting: A private infertility clinic. Patient(s): Women undergoing infertility treatment. Intervention(s): Metaphase II stage bovine and MII human oocytes underwent first polar body biopsy before cryopreservation in different vitrification conditions, and human oocytes were fertilized by intracytoplasmic sperm injection after warming. The resulting embryos were transferred into women undergoing infertility treatment. Main Outcome Measure(s): Postvitrification morphologic survival, in vitro blastocyst development, and clinical outcome after ET. Result(s): The equilibration temperature had a significant effect on cryosurvival of both bovine and human oocytes. High (97%–99%) postvitrification survival was achieved for both MII bovine and human oocytes, and high fertilization (90%–97%) at 35 C to 37 C, blastocyst development (18%–45%), and pregnancy (50%) rates were achieved at 35 C with 5.0 mol/L ethylene glycol þ 1.3 mol/L dimethyl sulfoxide for MII human oocytes that underwent first polar body biopsy. Conclusion(s): Previtrification equilibration temperature had a profound effect on the postthaw developmental competence of MII human oocytes in vitro and in vivo. The CryoLoop vitrification of first polar body–biopsied MII human oocytes in the presence of 5 mol/L ethylene glycol plus 1.3 mol/L dimethyl sulfoxide gave the best results in terms of fertilization, embryo development, and implantation rates. (Fertil Steril 2009;92:1706–15. 2009 by American Society for Reproductive Medicine.) Key Words: CryoLoop, cryopreservation, oocyte, zona slitting, vitrification

Long-term preservation of gametes, embryos, or gonadal tissues that contain an earlier form of gametes in conjunction with currently available assisted reproductive technologies presents tremendous opportunities for couples having infertility problems. In addition, preimplantation genetic diagnosis using polar body biopsy has become a valuable component of assisted reproductive technologies for preventing autosomal dominant disorders such as X-linked dominant diseases in women, as well as recessive disorders (1–5). Recently, there has been an increasing effort to cryobank human oocytes similar to sperm cryobanking, which has helped thousands of infertile couples. These efforts stemmed from a number of reproductive reasons including the desire to Received May 8, 2008; revised August 16, 2008; accepted August 21, 2008; published online October 20, 2008. L.K. has nothing to disclose. Y.A. has nothing to disclose. G.S. has nothing to disclose. M.K. has nothing to disclose. G.M. has nothing to disclose. Reprint requests: Levent Keskintepe, Ph.D., Sher Institute for Reproductive Medicine, 5320 S. Rainbow, Suite 300, Las Vegas, NV 89118 (FAX: 702-892-9666; E-mail: [email protected]).

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overcome infertility problems, to restore the fertility of women who will be undergoing chemotherapy or radiation therapy because of cancer treatment, and the desire for family planning. Under normal conditions preimplantation genetic screening has to provide an accurate outcome in a short time so that the resulting embryos can be transferred within the limited time window. The first polar body biopsy for preimplantation genetic screening and subsequent cryopreservation of biopsied metaphase II (MII) oocytes not only would allow an indefinite time window to complete the analysis but also significantly reduces the risk of misdiagnosis because it allows sufficient time to replicate the analysis in questionable situations. Until recently despite the significant efforts of numerous research groups, routine human oocyte cryopreservation remained an elusive task for human reproductive medicine. Because new vitrification technology has provided a sea change in outcome results (5), to date, oocyte cryopreservation still is

Fertility and Sterility Vol. 92, No. 5, November 2009 Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.

0015-0282/09/$36.00 doi:10.1016/j.fertnstert.2008.08.114

far less successful for nearly all mammalian species, with the exception of the mouse, in comparison with sperm and the preimplantation embryo (6). Research conducted over the past decades has demonstrated clearly that there are fundamental reasons for the susceptibility of mammalian oocytes to cryopreservation-induced injury, which prevents normal fertilization, as well as developmental progression to preimplantation stage embryos, including their sensitivity to cryoprotective agents and suboptimal conditions during the cryopreservation procedure (7). The studies attributed the poor cryosurvival of mammalian oocytes to some inherent factors including developmental stage and quality (8). In addition, it has been reported that mammalian oocytes are exceptionally sensitive to suboptimal conditions even before they are exposed to subzero temperatures. They are affected detrimentally by chilling, cryoprotective agents, and osmotic injury during addition of hyperosmotic cryoprotective agents. It also has been reported that the fertilization may be compromised by the cryoprotective agent concentration, its type, and temperature at which cryoprotective agents are introduced (9). A number of reports verified that increased concentrations of commonly used cryoprotective agents have toxicity to oocytes and a negative effect on the subsequent development of oocytes and embryos (10–12). There are reports that showed harmful effects of cooling on ultra structures of oocytes such as depolymerization of the meiotic spindle (8, 12, 13–15). To date, efforts have been focused on improving cryosurvival of mammalian oocytes (16). Conventional (slow) freezing procedure has not been accepted broadly for the purpose of mammalian oocyte cryopreservation including human oocytes because of chilling and freezing injury, but several other methods recently have been proposed to avoid the adverse effects of chilling and lethal ice formation during cooling (17–19). In essence, these protocols use very small amounts (1–3 mL) of vitrification solution to facilitate rapid cooling when the samples are plunged into liquid nitrogen (LN2). So-called minimum drop vitrification systems have allowed breakthrough results with bovine and porcine oocyte cryopreservation (20). So far, other vitrification techniques have been reported, and these methods used a few microliters of vitrification solution loaded into open pulled plastic straws (21), CryoLoops (Hampton Research, Aliso Viejo, CA) (18), and Cryotop (Kitazato, Japan) (22). Although availability of human oocytes for research is very limited, bovine oocytes share cryobiologically similar properties, therefore making bovine oocytes an excellent model to test various cryobiologically relevant interventions before testing on human oocytes. Both cell types are particularly sensitive to chilling and have a small surface-to-volume ratio. It has been suggested that a limiting factor for achieving cryopreservation of bovine oocytes is direct chilling injury (23) and osmotic damage (7), which take place during addition and removal of vitrification solution, and exposure to subphysiologic temperatures. Direct chilling injury, or cold shock, is defined as an irreversible damage expressed shortly Fertility and Sterility

after exposure to low, but not freezing, temperatures. Because the primary target for direct chilling injury and osmotic injury is the plasma membrane, we initially conducted preliminary studies on bovine oocytes to test the effect of zona slitting and vitrification temperature on the plasma membrane integrity. We subsequently determined the effect of different cryoprotective agents on MII human oocyte vitrification and previtrification temperature during cryoprotective agents’ duration of equilibration for the outcome of human oocyte vitrification. MATERIALS AND METHODS All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise stated. Bovine Oocytes In vitro matured bovine oocytes were purchased from Bomed, Inc. (Madison, WI). The zona pellucida of all oocytes was cleaned of all surrounding cells with a brief (2- to 3-minute) vortexing in 80 IU hyaluronidase/mL. The oocytes then were placed in 10 mL of modified human tubal fluid (mHTF) þ 10% synthetic serum supplement (Irvine Scientific, Irvine, CA), and maturation was graded. With use of a laser (Zilos-tk; Hamilton Thorne Bioscience, Beverly, MA), a slit was made on the zona pellucida directly over the polar body traversing the perivitelline space (Fig. 1). Human Oocytes Human oocytes were retrieved from 21 oocyte donors who were younger than 35 years of age (mean 29.4  4.3 years) under the surveillance of the Western Institution Review Board. In addition, 10 patients undergoing IVF (mean age 34.3  1.7 years) were included for this study. They all underwent comprehensive physical examinations and standard Food and Drug Administration–required laboratory testing. Ovarian stimulation, oocyte retrieval, and polar body biopsy After appropriate disclosure, each patient underwent controlled ovarian hyperstimulation as previously described (24). Human chorionic gonadotropin, 10,000 IU (Profasi; Organon Pharmaceuticals, Kenilworth, NJ) was used to trigger ovulation in all cases. Transvaginal needle-guided oocyte retrievals were performed 34 to 37 hours later with use of IV propofol to induce conscious sedation. The surrounding cumulus oocyte complexes were stripped from each MII oocyte and graded, and polar body biopsy was performed as previously described (4). Metaphase II oocytes thereupon were vitrified individually with use of the CryoLoop system described by Mukaida et al. (25). Vitrification of MII oocytes All MII oocytes were cultured at 37 C in mHTF (IVF Online, Guelph, Canada) supplemented with 10% synthetic serum supplement until vitrification. Metaphase II oocytes were placed consecutively in different aliquots of vitrification solution, each comprising a different concentration of mHTF þ dimethyl sulfoxide (DMSO) 1707

FIGURE 1 Intracytoplasmic sperm injection and subsequent blastocyst development of polar body–biopsied and vitrifiedwarmed MII human oocytes.

Keskintepe. Oocyte cryopreservation after polar body biopsy. Fertil Steril 2009.

(D2650; Sigma) (i.e., 0.13, 0.28, 0.64, 0.95, 1.9 mol/L) þ ethylene glycol (EG) (E 9129; Sigma) (i.e., 0.16, 0.35, 0.8, 1.2, 2.4 mol/L) for 30 seconds, 30 seconds, 1 minute, 1 minute, and 90 seconds, respectively. Finally, each MII oocyte was transferred to a vitrification solution (in mHTF) comprising 2.6 mol/L DMSO þ 3.3 mol/L EG þ 1.0 mol/L sucrose (S1888; Sigma) þ 0.1 mol/L Ficoll (PM70; Amersham Biosciences, Piscataway, NJ), for 30 seconds and then loaded in a 0.5 mm CryoLoop. They then were plunged directly into LN2 and placed in 1 mL plastic vials for long-term storage. Experiment 1 The effects of equilibration temperature, zona slitting by laser, and subsequent polar body biopsy on bovine oocyte viability after vitrification were investigated. Five experimental groups were tested: [1] control (no treatment) at 37 C; [2] zona slitting and vitrification at 37 C; [3] zona slitting, first polar body biopsy, and vitrification at 37 C; [4] zona slitting and vitrification at 22 C; and [5] zona slitting, first polar body biopsy, and vitrification at 22 C. A total of four replications were performed for each group. All MII bovine oocytes were vitrified in 2.6 mol/L DMSO þ 3.3 mol/L EG (ED20). After 24 to 48 hours storage in LN2, they were warmed as follows: Individual CryoLoops were removed from their plastic vials and plunged into media containing 1.5 mol/L sucrose at 37 C and held for 50 seconds. They then were placed sequentially in sucrose, 1.5 mol/L to 1.0, 1708

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0.75, 0.5, 0.25, 0.125, and 0.0 mol/L respectively, for 0.5 to 2 minutes in each concentration. Assessment of plasma membrane integrity An inverted microscope (Nikon, Melville, NY) was used to monitor (with 20 objective) hyperosmotically induced cell dehydration and subsequent rehydration after exposing MII bovine oocytes to 0.5 mol/L sucrose within 2 and 24 hours of thawing. The oocytes were monitored until complete shrinkage took place on exposure to 0.5 mol/L sucrose solution. They then were suspended into isosmotic mHTF solution at 37 C, and time required to gain isosmotic volume was recorded. Bovine oocytes were cultured individually in Global One Medium (IVF Online) with 10% synthetic serum supplement (Irvine Scientific) for 2 and 24 hours after warming. Experiment 2 Metaphase II human oocytes were assigned randomly to the following experimental groups: [1] 6.7 mol/L ethylene glycol (E40); [2] 3.3 mol/L EG þ 2.6 mol/L DMSO (ED20); and [3] 5.0 mol/L EG þ 1.3 mol/L DMSO (ED30). Oocyte manipulations were carried out at either 37 C or 35 C. After warming, oocyte recovery was recorded, and those oocytes that survived were fertilized by intracytoplasmic sperm injection (ICSI) to examine the fertilization and embryonic development as blastocyst formation as an end point.

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Experiment 3 To determine developmental competence of MII human oocytes that were cryopreserved with use of different vitrification conditions at experiment 2, we transferred the blastocysts subsequently into the designated patient, and the pregnancy and implantation rates were recorded. Intracytoplasmic Sperm Injection and ET Progesterone injections were initiated 6 days before planned ET, and vitrified oocytes were sequentially warmed one at a time until a total of three viable MII oocytes were available. Once oocytes were warmed and viability confirmed, they were kept in culture for 2 hours before ICSI. Intracytoplasmic sperm injection was accomplished by introducing a single sperm-containing 30 degree angled ICSI needle (Humagen, Charlottesville, VA) from the 3 o’clock position when the polar body was stabilized at either the 12 or 6 o’clock position (Fig. 1). Presumptive zygotes and cleaved embryos were cultured individually to the blastocyst stage in Global One (IVF Online) media at 37 C, in an environment of 6% CO2, 5% O2, 89% nitrogen, and 95% humidity. Only those embryos that had developed to the expanded blastocyst stage by day 5 or 6 after ICSI and exhibited well-developed inner cell masses and trophectoderms were deemed eligible for ET. Preparation of Embryo Recipients Recipients were subjected to physical and emotional criteria that were determined through a detailed medical history, physical examination, psychological examination, and laboratory testing of both prospective parents. Full disclosure was made to all recipient couples, who were also counseled and thereupon were required to sign a written consent form. All women had regular uterine cavities as assessed by preceding sonohysterography or hysteroscopy and had preceding ultrasound evidence of endometrial linings that measured R9 mm around the time of spontaneous or induced ovulation. Hormonal Treatment of Embryo Recipients An E2 valerate (4–8 mg IM) was administered every 3 days starting with the onset of birth control pill–induced menstruation until the plasma [E2] was stabilized. The goal of stabilizing the plasma [E2] at 500 to 1,000 pg/mL in association with an endometrial thickness of R9 mm was achieved within 8 to 12 days in all cases. Thereupon, daily IM injections of 100 mg P in oil were initiated. At the same time previously vitrified oocytes were warmed so as to obtain three viable oocytes available for ICSI with partners’ or designated donor sperm. The fertilized oocytes were cultured for up to 6 days as described. Embryo Transfers Subject to patient choice and availability, up to two blastocysts were transferred in each case. Leftover blastocysts were revitrified and cryobanked for future dispensation at Fertility and Sterility

the sole discretion of the designated recipient and her partner. Embryo transfers were performed on the sixth day of progesterone in oil administration (day 5 of embryo development to the blastocyst stage). In the event of pregnancy, the prescribed estrogens-progesterone regimen was continued to the 10th week of gestation whereupon it was discontinued abruptly. In all cases where pregnancy did not occur, or failed to survive, hormonal treatment was stopped immediately. Statistical Analysis Data with regard to postvitrification survival, rehydration and dehydration time, fertilization, and embryonic development were analyzed by use of analysis of variance with the Sigma STAT 3.0 statistical package (Systat Software Inc., San Jose, CA). The data on pregnancy rates were analyzed by c2 test. RESULTS Experiment 1 The results of experiment 1 are presented in Table 1. The MII bovine oocytes that were treated at 37 C before vitrification had shorter dehydration and rehydration time than those equilibrated at 22 C (P<.05). A similar trend with regard to dehydration and rehydration time was also observed after 24-hour in vitro cultured oocytes. The differences in volume responses of the oocytes was correlated with postvitrification survival in that equilibration of MII bovine oocytes in ED20 vitrification solution at 37 C resulted in superior (P<.05) cryosurvival over those treated at 22 C. Metaphase II bovine oocytes that were equilibrated in ED20 vitrification solution at 22 C resulted (87%) in significantly lower (P<.05) cryosurvival than for those treated at 37 C (97%). Similarly, the polar body biopsy of MII bovine oocytes that were equilibrated in ED20 vitrification solution at 22 C (86%) resulted in lower cryosurvival than for their counterparts equilibrated at 37 C (98%). Experiment 2 The cryosurvival, post-ICSI fertilization, and in vitro blastocyst development rates of MII human oocytes that were equilibrated in vitrification solution at 22 C for ED20 (96%, 94%, and 18%), ED30 (93%, 91%, and 16%), and E40 (89%, 88%, and 13%), respectively, are given in Table 2. The corresponding cryosurvival, post-ICSI fertilization, and in vitro blastocyst development rates of MII human oocytes that were equilibrated in the same vitrification solutions at 37 C for ED20 (98%, 92%, and 18%), ED30 (99%, 97%, and 37%), and E40 (97%, 90%, and 47%) and at 35 C for ED20 (99%, 91%, and 27%), ED30 (97%, 93%, and 33%), and E40 (97%, 97%, and 45%), respectively, were given. Although there was a statistical difference (P<.05) among the groups for cleavage rates on day 3 at the equilibration temperature of 22 C (Table 2), the blastocyst development rates did not differ (P>.05). There was no difference among the treatment groups with regard to postthaw survival and fertilization rates regardless of 1709

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TABLE 1 Postthaw recovery and duration of dehydration and rehydration of MII bovine oocytes after being subjected to 0.5 mol/L sucrose solution and subsequently returned to isosmotic condition in 2 or 24 hours on recovery.

Groups 1. Control 2. ZP slitting 3. ZP slitting and PBx 4. ZP slitting 5. ZP slitting and PBx

No. oocytes No. oocytes No. oocytes Dehydration Rehydration Dehydration Rehydration Equilibration vitrifieda and recovered responded time (min) time (min) 2 h time (min) 24 h time (min) 24 h warmed temperature (%) (%) 2 h after thaw after thaw after thaw after thaw 37 C 37 C 37 C 22 C 22 C

Control 110 100 110 110

N/A 103 (94) 96 (96) 110 (100) 107 (98)

100 (100)b 100 (97)b 94 (98)b 96 (87) 92 (86)

0:55  0:15b 1:00  0:20b 1:00  0:20b 2:05  0:15b 2:25  0:05b

3:50  0:25 3:25  0:30 3:25  0:30 4:50  0:30 4:55  0:35

Note: Dehydration and rehydration were performed at 37 C. NA ¼ not applicable; ZP ¼ zona pellucida; PBx ¼ polar body biopsy. a Vitrification was performed in ED20 solution (2.6 mol/L DMSO þ 3.3 mol/L EG). b Statistically different at P<.05 compared with groups 4 and 5. Keskintepe. Oocyte cryopreservation after polar body biopsy. Fertil Steril 2009.

1:05  0:10b 1:15  0:15b 0:55  0:25b 1:25  0:25b 1:20  0:30b

3:50  0:30b 3:25  0:30b 3:25  0:30b 4:00  0:20b 3:55  0:35b

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TABLE 2 Effect of equilibrating MII human oocytes in various vitrification solutions at 37 C on their postthaw recovery, fertilization, and blastocyst development. Equilibrating temperature for vitrification 22 C ED20 No. oocytes vitrified No. oocytes thawed No. oocytes survived (%) No. oocytes injected No. oocytes fertilized (%) No. embryos cleaved to 6> cell (%) No. blastocysts on day 5

ED30

35 C E40

ED20

ED30

37 C E40

ED20

ED30

E40

96

82

86

64

57

56

86

81

82

96

82

86

64

57

56

86

81

82

92 (96)

76 (93)

82 (89)

63 (99)

55 (97)

54 (97)

84 (98)

80 (99)

79 (97)

92

76

82

63

55

54

84

80

79

86 (94)

69 (91)

72 (88)

57 (91)

51 (93)

52 (97)

77 (92)

77(97)

71 (90)

19 (22)a

23 (34)

13 (18)a

18 (32)b

21 (39)b

28 (54)

18 (24)a

34 (45)

23 (26)a

15 (18)

11 (16)

9 (13)

17 (27)

18 (33)

18 (35)

14 (18)c

28 (37)

19 (27)c

Note: ED20 ¼ 2.6 mol/L DMSO þ 3.3 mol/L EG; ED30 ¼ 1.3 mol/L DMSO þ 5.0 mol/L EG; E40 ¼ 6.7 mol/L EG. a Statistical significance at P<0.05 compared with ED30. b Statistical significance at P<0.03 compared with E40. c Statistical significance at P<0.03 compared with ED30. Keskintepe. Oocyte cryopreservation after polar body biopsy. Fertil Steril 2009.

equilibration temperature (P¼.05). However, the cleavage rates (more than six cells) on day 3 and the blastocyst development rates on day 5 were significantly higher (P<.05) for ED30 (45% and 37%) than those in ED20 (24% and 18%) and E40 (26% and 27%) at the equilibrium temperature of 37 C. When equilibration temperature was lowered from 37 C to 35 C, however, cleavage (more than six cells) on day 3 significantly increased (P<.05) for oocytes that were equilibrated in E40 (54%) over those in E20 (32%) and E30 (39%). Experiment 3 Table 3 summarizes the confirmed pregnancy and implantation rates of vitrified MII human oocytes after ET. Equilibration of MII human oocytes in ED30 vitrification solution at 35 C provided a higher (80%) pregnancy rate than those oocytes that were equilibrated in ED20 vitrification solution at 22 C (50%) or in ED30 vitrification solution at 37 C (43%). However, implantation rates of blastocysts that were developed from human oocytes equilibrated in ED30 at 35 C before vitrification were significantly (P<.05) higher (50%) than those equilibrated in ED20 solution at 37 C (38%) or at 22 C (25%). Fertility and Sterility

DISCUSSION We previously reported successful karyotyping of human oocytes after polar body biopsy by comparative genomic hybridization in an effort to select ‘‘competent’’ embryos and significantly improved IVF outcome (4). In this study, further studies were performed to incorporate cryopreservation of polar body–biopsied oocytes to take full advantage of time required for reliable genetic testing and also provide future reproductive options to women or couples who would like to have infants. To this end, the overall goal of the present study was to improve cryosurvival of MII human oocytes after laser zona slitting and polar body biopsy with use of a CryoLoop vitrification technique. To achieve our longterm goal we first evaluated the effect of previtrification equilibration temperature, laser-assisted zona slitting, and vitrification solution on cryosurvival of MII bovine oocytes and subsequently used this information in an effort to improve cryosurvival of MII human oocytes. During the cryopreservation procedure, the oocyte undergoes dramatic osmotically induced volume changes due to hyperosmotic freezing solutions, which cause sudden water and cryoprotective agent transport across the oolemma (25). Furthermore, the temperature at which the cells are 1711

TABLE 3 Pregnancy result of MII human oocytes that were vitrified in different conditions. ED20 at 22 C

ED20 at 37 C

ED30 at 35 C

No. of patients 8 7 5 No. oocytes 56 63 25 vitrified No. oocytes 24 21 15 thawed No. oocytes 23 (96) 19 (91) (100) survived (%) No. oocytes 23 19 15 injected No. oocytes 18 (79) 17 (90) 14 (94) fertilized (2PN) No. oocytes 6 (34)a 5 (30)a 8 (58) developed to >6 cells (%) No. blastocysts 1.7  0.3 1.7  0.4 1.5  0.5 on day 5 No. of live 4 (50)b 3 (43)b 4 (80) births (%) No. of singleton 3 3 2 No. of twin 1 0 1 No. of 5/13 (38)c 3/12 (25)c 4/8 (50) implantations/ embryos (%) Note: ED20 ¼ 2.6 mol/L DMSO þ 3.3 EG; ED30 ¼ 1.3 mol/L DMSO þ 5.0 mol/L EG. 2PN ¼ two pronuclei. a P<0.05 compared with ED30 at 35 C. b P<0.05 compared with ED30 at 35 C. c P<0.05 compared with ED30 at 35 C. Keskintepe. Oocyte cryopreservation after polar body biopsy. Fertil Steril 2009.

exposed to these cryopreservation solutions has different osmotic and toxic consequences on cell cryosurvival because of temperature-dependent hydraulic conductivity, cryoprotective agent permeability, and disintegration of the meiotic spindle and microfilaments in the ooplasm (8). In this context, some of the previously determined membrane permeability characteristics such as cryoprotective agents and their temperature-dependent permeability coefficient would provide useful information with regard to cell membrane behavior in the presence of various cryoprotective agents at different temperatures (26). One could use the information gathered from a particular cell type and species (i.e., bovine oocyte) to have useful models in an effort to improve cryosurvival, if both cell types behave similarly when they undergo similar stress factors. Although differences exist between human and bovine oocytes (e.g., lipid content of the bovine oocytes is higher than that of human oocytes) there are also remarkable similarities such as extreme chilling sensitivity, cell surface-to-volume ratio (diameter ranging from 110 to 1712

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130 mm), and membrane water and cryoprotective agent permeability coefficients (27–29). Activation energy for DMSO and EG was calculated to be 20.80 kcal/mol and 21.78 kcal/ mol for MII human oocytes (28–30). Interestingly, the corresponding estimated value for MII bovine oocytes is 21.0 kcal/ mol (31). Thus, we selected MII bovine oocytes to establish a base for MII human oocytes that have undergone laser-assisted slitting and polar body biopsy before their cryopreservation via vitrification technique. The oocytes need to maintain the integrity of several structural features to undergo fertilization and development after cryopreservation. However, it has been reported that MII spindle fibers and cortical granules are affected adversely by subphysiologic temperatures (19). For example, oocyte microtubule depolymerization begins even with a fall in temperature of only 5 C in humans and seems irreversible when oocytes are exposed to 27 C, and this effect worsens as duration of chilling is prolonged (8, 15). In addition, it was previously reported that cryoprotective agents such as DMSO and EG cause premature induction of cortical granule exocytosis and subsequent zona hardening (18, 32–34). We overcame this problem by using ICSI procedure in the current study. An early study with MII mouse oocytes, while showing that chilling causes MII spindle depolymerization, also demonstrated the ability of the MII spindles to undergo repair at a very high rate after slow cooling in the presence of DMSO (35, 36, 37). Dimethyl sulfoxide also might be effectual in protecting the meiotic spindle and other structures of the porcine oocyte during vitrification (38). This phenomenon of the mouse oocyte may offer a partial explanation for higher cryosurvival of MII mouse oocytes compared with those of other species. It was proposed that with the appropriate exposure to cryoprotective agents, optimal slow cooling and thawing rates would allow MII human oocytes cryopreservation without significant changes in their second meiotic spindle organization and without increase in the rate of aneuploid embryos (39). If performed appropriately, ultrarapid (approximately 25,000 C/min) cooling so-called vitrification would significantly eliminate formation of lethal ice crystals and protect the oocyte from chilling injury (40), which would be observed during conventional freezing. It should be noted that the vitrification technique also has the shortcoming of an increased probability of other form of injuries such as cryoprotective agent toxicity, oolemma rupture, and microtubule and filament disruption due to relatively high (6–7 mol/L) cryoprotective agent concentration (29). Different approaches have been proposed to minimize these adverse effects such as use of relatively less-toxic chemicals, combination of two or three cryoprotective agents, and stepwise addition and/or exposure of cells to precooled concentrated solutions (41, 42) as it was done in the present study. Cell plasma membrane is one of the most sensitive structures of the oocyte particularly during exposure to extremely hyperosmotic vitrification solutions. In the present study, we used a stepwise addition and dilution process

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to avoid drastic volume-affecting adverse changes. Cell plasma membrane integrity after cryobiologically relevant stressors has been evaluated widely by fluorescent techniques including carboxyfluorescein–propidium iodide (43) and SYBR-14–propidium iodide (44). Intact plasma membrane consists of semipermeable lipid bilayers and has the ability to respond to surrounding osmotic alterations accordingly. Cells with intact plasma membrane are expected to swell because of water influx when they are exposed to hyposmotic solution, whereas cells are expected to shrink because of dehydration when they are subjected to hyperosmotic condition. In addition, cells cannot regain their original shape and volume when they are exposed continuously to nonpermeating ionic (NaCl) or nonionic (sucrose) solutes unless they are returned to appropriate isosmotic culture conditions. In this study, we successfully used these osmotic properties of cells to evaluate the plasma membrane integrity of MII bovine oocytes after polar body biopsy and vitrification. This study demonstrated that oolemma integrity of MII bovine oocytes was highly (97%–98%) protected after they underwent first polar body biopsy and vitrification in the presence of 2.6 mol/L DMSO and 3.3 mol/L EG at 37 C. Significant membrane integrity loss (15%) took place when equilibration in vitrification solution was performed at 22 C. In addition, the membrane integrity of these MII bovine oocytes was maintained even after 24 hours in vitro culture, suggesting their robustness in terms of long-term developmental potential after first polar body biopsy and vitrification. It was reported that the temperature at which the cells are equilibrated is one of the most important causes of the reduced developmental competence of vitrified bovine oocytes (10). The current results supported this early study in that it is better for MII bovine oocyte cryosurvival if equilibration is performed at 37 C rather than 22 C. The lower survival of oocytes that were equilibrated in ED20 at 22 C before vitrification may be explained by a lesser amount of intracellular cryoprotective agent concentration due to a lower cryoprotective agent permeability coefficient resulting in insufficient vitrification (26, 30). Equilibrating first polar body–biopsied MII bovine oocytes in ED20 at 22 C before vitrification resulted in lower postthaw morphologic survival than for those treated at 37 C. However, this difference in terms of pregnancy rate was diminished (50% vs. 43%) when these two vitrification conditions were used to vitrify first polar body–biopsied MII human oocytes. This suggests that although initial survival is an essential step forward for cell survival there are many other factors that cumulatively affect oocyte developmental competence during later stages of development. In this study, equilibrating human oocytes at 35 C resulted in higher blastocyst formation rate than equilibrating at 37 C in ED20 (27% vs. 18%) or E40 (45% vs. 27%) but was comparable in ED30 (33% vs. 37%). It may be possible that lowering equilibration temperature a few degrees to 35 C provides intracellular cryoprotective agent concentration that is suffiFertility and Sterility

cient to achieve vitrification, as well as reducing the chemical toxicity of cryoprotective agents. We also should be reminded that commonly used vitrification solutions are generally complex because they contain different types and concentrations of cryoprotective agents, sugars, macro molecules, and numbers of other biologic or synthetic components (i.e., serum). It is often difficult to determine the actual effects of each compound on the cell because of potential temperature-dependent interactions from each other. Thus, although it is premature to make a general conclusion with regard to the best vitrification conditions because of relatively limited numbers of ETs in this study, use of ED30 vitrification solution at 35 C provided a superior pregnancy rate (80%) as compared with ED20 vitrification solution at either 22 C or 37 C. In a recent study, by exposing MII human oocytes to combined vitrification solutions of EG (15%), DMSO (15%), and sucrose (0.6 mol/L) at 25 C, Selman et al. (45) achieved 75% (18/24) morphologic survival and 78% fertilization rate (14/18). After transfer of resulting embryos to six patients (2.3 embryos per patient) they obtained two pregnancies (33%): one singleton and one twins. They equilibrated MII human oocytes in EG þ DMSO–based vitrification solution at 25 C. This temperature is similar to one of our treatment groups, ED20, which also used 22 C during equilibration. Although our postthaw survival rate is higher (96%) than theirs (75%), fertilization rates between two studies were very close (78% vs. 79%). On the other hand, the pregnancy rate in our study (50%) is much higher than in their study (33%) although we used 1.7 embryos per patient in our study as compared with 2.3 embryos per patient in their study. Preimplantation genetic diagnosis has become one of the useful components in assisted reproductive technologies because of advanced maternal age–related aneuploidy and X-linked diseases (1). In this study, we successfully demonstrated feasibility of human oocyte polar body biopsy and subsequent vitrification. The current data further suggest that physical intrusions such as laser-assisted zona slitting and polar body biopsy before vitrification do not significantly affect morphologic survival of MII bovine oocytes after cryopreservation regardless of vitrification condition used. It should be noted, however, that there may have been subcellular damages that we have not investigated in detail in the present study for bovine oocytes with the exception of membrane integrity. On the other hand, MII human oocytes that underwent the same physical interventions have resulted not only in high (97%–99%) morphologic survival but also in high fertilization (91%–97%) and development to the blastocyst stage (up to 45%) after ICSI. These data suggest that the whole vitrification procedure including zona slitting before vitrification had little effect on the developmental competence of MII human oocytes in vitro. However, it should be stressed that in vivo development competence of embryos that were produced from oocytes that underwent first polar body biopsy and vitrification is the most essential measure for the broad acceptance of the procedures described in this

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study. We therefore proceeded with ETs in designated patients. The clinical outcome of the initial studies was consistent with the in vitro results in that as high as a 50% pregnancy rate can, we believe, be achievable clinically in infertility programs. There have been limited animal studies describing polar body biopsy of oocytes or pronuclear stage embryos in combination with cryopreservation, although Isachenko et al. (44) were successful in the mouse. This study provided the proofof-principle with use of first polar body biopsy and subsequent vitrification in MII bovine oocytes. Very recently, Naether et al. (5) reported pregnancy after transfer of embryos that were derived from vitrified pronuclear stage human embryos that underwent first and second polar body biopsies for aneuploidy screening. However, to our best knowledge this is one of the first reports of high (50%) pregnancy after vitrification of MII human oocytes that underwent polar body-1 biopsy for genetic screening. In conclusion, the present data overall suggest the possibility of using bovine oocytes as an initial testing ground when designing human oocyte cryopreservation protocols. Most important, successful first polar body biopsy of MII human oocytes in combination with the cryopreservation procedure described in this study would be very helpful to improve human reproductive and public health in general. Our current effort is being directed to larger numbers of ETs to further support our claim. Acknowledgments: The authors thank Simon Fishel, Ph.D., for his critical review of the manuscript.

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