Middle East Fertility Society Journal (2011) 16, 189–193
Middle East Fertility Society
Middle East Fertility Society Journal www.mefsjournal.com www.sciencedirect.com
ORIGINAL ARTICLE
Comparison of two different media for vitrification and rewarming of human zygotes: Prospective randomized study Ebru Alcolak a,b, Ehab Abu Marar a,b, Sotiris C. Mytas Vassili Palapelas c, Beate Scho¨pper a,b, Klaus Diedrich a b c
a,b
, Nectarios Chalvatzas a,b, a,b , Safaa Al-Hasani a,b,*
University of Schleswig-Holstein, Department of Gynaecology and Obstetrics, Ratzeburger Allee 160, 23538 Lu¨beck, Germany Reproductive Medicine Unit, University of Lu¨beck, Ratzeburger Allee 160, 23538 Lu¨beck, Germany Department of Obstetrics and Gynaecologic Clinic, Aristotle University of Thessaloniki, Greece
Received 18 December 2010; accepted 23 February 2011 Available online 20 August 2011
KEYWORDS Vitrification; Home-made vitrification media; Commercial vitrification media
Abstract Vitrification has been successfully used for cryopreservation of human oocytes, zygotes and embryos. There are various media which contain different combinations and concentrations of cryoprotectants for this technique. In this prospective randomized study, two different vitrification and warming media were compared in human zygotes’ cryopreservation. Ó 2011 Middle East Fertility Society. Production and hosting by Elsevier B.V. All rights reserved.
1. Introduction Recent publications show that the implantation and pregnancy rate are higher in frozen-warmed embryo transfer after vitrification than in fresh embryo transfer (1). New studies indicated that using cryopreserved-warmed rather than fresh embryos reduces the risk of stillbirth and preterm delivery (2–4). This * Corresponding author at: University of Schleswig-Holstein, Department of Gynaecology and Obstetrics, Ratzeburger Allee 160, 23538 Lu¨beck, Germany. Tel.: +49 4515002155; fax: +49 4515004746. E-mail address:
[email protected] (S. Al-Hasani). 1110-5690 Ó 2011 Middle East Fertility Society. Production and hosting by Elsevier B.V. All rights reserved. Peer review under responsibility of Middle East Fertility Society. doi:10.1016/j.mefs.2011.02.003
Production and hosting by Elsevier
might be due to a better quality of embryos surviving after the freeze-warm process or to a better implantation window at the time of implantation. It is clear that cryopreservation of both human oocytes and zygotes became more valuable in assisted reproductive technology (ART) since the publication of the first reports about biochemical and successful clinical pregnancies with frozen-warmed human embryos in the early 1980s (5). Vitrification is an ultra-rapid method of cryopreservation that has been widely used in many in vitro fertilization (IVF) centers. High concentration of cryoprotectants with rapid cooling rates is essential to cryopreserve embryos as well as oocytes in a vitrified, glass-like state (6). Therefore, it indicates that there is a strong correlation between concentration of cryoprotectant and cooling rates during vitrification. However, a higher concentration of cryoprotectants which may cause toxic and osmotic effects has been a main trouble limiting its popularity since its introduction in mid-1990s (7). Also during vitrification, since a very high concentration of cryoprotectant is used, it is preferable to expose the embryos and oocytes to the cryoprotectant for a short period to avoid its toxic effect.
190 Vitrification of human embryos and oocytes is possible with different cryoprotectants media, such as dimethyl sulfoxide (DMSO) or propanediol (PrOH) in combination with ethylene glycol (EG), or EG alone (8–10) It has also been reported that cryoprotectant mixtures might have better results than the media containing one permeable cryoprotectant (11). The combination of EG and DMSO has been successfully used (12–14). Kuwayama et al. described a successful vitrification media consisting of 15% EG, 15% DMSO and 0.5 mol/l sucrose and two media used for warming with a sucrose concentration of 1.0 and 0.5 mol/l, respectively (15). Jelinkova et al. described vitrification media by using EG and trehalose for 2PN human zygotes (16). Vanderzwalmen et al. showed that vitrification procedure using a medium with EG, sucrose and ficoll as cryoprotectants is simple and efficient for the cryopreservation of day 4 morulae or day 5 early blastocysts (17). We designed this prospective randomized study to compare between two different vitrification and warming media in human zygotes in IVF/intracytoplasmic sperm injection (ICSI) cycles and this is the first comparison of EG–DMSO with EG–PrOH for human zygotes. 2. Materials and methods 2.1. Patients This study consisted in total of 63 cycles of cryopreserved embryo transfer and vitrification of 2PN stage zygotes of 64 patients who underwent IVF/ICSI at the Reproductive Medicine Unit, Department of Gynaecology and Obstetrics, University of Lu¨beck, Germany between April 2008 and March 2009. All patients enrolled in this study were informed and gave a written consent for the cryopreservation procedure. The mean age of all patients is 33, 34 (±4, 16) years and the cause of infertility was either male factor infertility or a combination with tubal infertility. 2.2. Ovarian stimulation and oocyte retrieval Ovulation induction was performed by administration of gonadotropin-releasing hormone (GnRH) antogonist (Cetrotide, Serono, Germany) together with gonadotropins (Gonal F, Serono; Menopur, Ferring, Germany). Ovulation was triggered by the i.m. administration of human chorionic gonadotrophin (HCG) (10,000 IU/ml, Choragon, Ferring) as soon as three follicles of a diameter of P17 mm were observed by transvaginal ultrasonography and with estradiol concentrations corresponding to the number of follicles. Oocyte retrieval was performed 35–36 h after triggering under ultrasonographic guidance. Retrieved oocytes were transferred to dishes containing a culture medium (Sage Media), and were submitted to ICSI procedure as described by Al-Hasani et al. (18). 2.3. Vitrification and warming procedures Depending on the case, randomizing supernumerary PN stage zygotes was prepared for the vitrification procedure with either a solution that contains EG–PrOH (commercial media, Vitrolife) or a solution that contains EG–DMSO (home-made med-
E. Alcolak et al. ia). Vitrification procedure was performed at even days with home-made media (HM) and at non even days with commercial media (CM) starting day 1 after ICSI procedure. The vitrification-warming procedure was performed according to the method that was described by Kuwayama in HM (15). In this protocol, the 2PN stage zygotes were incubated in equilibration solution which contains 7.5% EG (Sigma–Aldrich, Steinheim, Germany) and 7.5% DMSO (Sigma–Aldrich) in Ham’s F-10 media supplemented with 10% human albumin (Sage) for 5–15 min (depending on the time needed for re-expansion of the cell) at room temperature. After an initial shrinkage and recovery, 2PN zygotes were aspirated and placed into vitrification solution containing 15% EG, 15% DMSO, 0.5 M sucrose (Merck, Darmstadt, Germany) in Ham’s F-10 medium supplemented with 10% of human albumin (Sage) for 50–60 s at room temperature. After that, zygotes were aspirated and placed on the tip of a Cryotop (Kitazato, Japan). In CM (RapidVitä Cleave, Vitrolife), vitrification was performed according to the introduction that was given by the company. In this protocol, we used three different media. Vitrification procedures were performed at 37 °C. The zygotes were transferred from the culture medium into the Vitri 1ä Cleave solution (MOPS buffered medium containing gentamicin and human serum albumin) for 5 min; Vitri 2ä (EG) Cleave solution for 2 min; Vitri 3ä Cleave solution (EG and PrOH) for 30 s. The zygotes were aspirated and placed on the tip of the Cryotop (Kitazato, Japan). No more than two zygotes were placed on each Cryotop. Cooling of the zygotes was performed by direct contact with liquid nitrogen in both protocols. The Cryotops were stored in liquid nitrogen for at least 1 month. In HM, warming of zygotes was performed by placing the Cryotop in a warming solution (1 M sucrose) for 50–60 s at 37 °C and moving into a dilution solution (0.5 M sucrose) for 3 min at room temperature. The warmed zygotes were placed 4–5 times into a washing solution (Cleavage-Medium, SAGE) before incubation. Warming procedure in CM was performed RapidWarmä Cleave, Vitrolife kit. All manipulations of the embryos were carried out at 37 °C. Warming Solution 1 (0.65 M sucrose) for 30 s; Warming Solution 2 (0.325 M sucrose) for 1 min; Warming Solution 3 (0.125 M sucrose) for 2 min; Warming Solution (0 M sucrose) for 5 min. The intact zygotes were cultured in a cleavage-medium for 24 h prior to embryo transfer. The embryo quality was evaluated comparing their morphology in both protocols. We also described the relative fragmentation that occured in the embryos. 0% fragmentation is called grade A, 0–20% is called grade B, 20–50% is called grade C, >50% is called grade D (19). 2.4. Embryo transfer and preparation of endometrium Patient’s endometrium was prepared initially by the administration of estradiol valerate (Progynova, Schering, Germany) (2 mg three times a day, initiated on the third day of the cycle). If pregnancy occured, it has been continued for 10 weeks. An endometrial thickness of 8 mm was considered optimal for performing embryo transfer and intravaginal administration of micronized progesterone (50 mg/day, Crinone gel, Serono) was done on the same day and 48 h prior to embryo transfer. A soft transfer catheter (Cook, USA) was used for embryo transfer on day 17 of the cycle.
Comparison of two different media for vitrification and rewarming of human zygotes: Prospective randomized study 3. Statistical analysis
191
Table 2 Embryo scoring of vitrified and warmed zygotes after a culture period of 24 h.
Data are shown as mean values ± SD, Pearson’s Chi-square test was used for analysis as appropriate P-value was considered statistically not significant. 4. Results In total, 128 2PN zygotes from 64 patients were vitrified during the study period and these vitrified zygotes were warmed in order to perform 63 transfer cycles. Of the included patients, 63 had one cycle of embryo transfer, one had no transfer cycle because the zygotes after warming did not divide. Table 1 describes the outcome of these transfer cycles for both vitrification protocols. The mean age of the patients was 32.92 (±4.10) and 33.64 (±4.23) for commercial kit and home-made media, respectively. Using CM, vitrified/warmed 2PN stage zygotes had a survival rate of 100% (n = 54/54), while for HM, it was 98.64% (n = 73/74; P = 0.3911). Commercial kit had a cleavage rate of 88.88% (n = 48/54) and for home-made media it was 97.26% (n = 71/73; P = 0.05489). Twenty-six cycles of embryo transfer were performed for commercial kit and the mean of transferred embryos was 1.84, for home-made media, 37 cycles of embryo transfer were performed with a mean of transferred embryos of 1.91. After 24 h of culture, 20 (41.66%) and 40 (56.33%) zygotes divided to 4-cell stage embryos in commercial and home-made media, respectively. The majority of cleaved embryos in both media had a quality score of grade A (Table 2). A positive serum hCG concentration was measured 14 days after embryo transfer in all patients. The implantation rate as well as the pregnancy rate was calculated for both media. Implantation rate for commercial kit was 14.58% (n = 7/48) and 14.08% (n = 10/71; P = 0.9392) was for home-made media. Pregnancy rate for CM was 26.92% (n = 7/26) and for HM 27.02% (n = 10/37; P = 0.9927). To summarize, of these 14 clinical pregnancies, 13 resulted in healthy live births.
Table 1 Major outcomes of the cycles of cryopreservation and embryo transfer for both protocols. Parameter
Commercial media
Home-made media
No. of patients No. of cycles of embryo transfer No. of 2PN vitrified/warmed zygotes No. of 2PN zygotes survived Survival rate (%) No. of cleaved embryos Cleavage rate (%) No. of 2PN discarded zygotes No. of 2PN arrested zygotes No. of transferred embryos Mean No. of embryos per transfer No. of positive hCG tests (%) No. of clinical pregnancies (%) Implantation rate (%) Incidence of twins Incidence of triplet No. of spontaneous abortions Live birth (%)
27 26 54 54 100 48 88.88 1 5 48 1.84 7 (26.92) 6 (23.07) 14.58 1
37 37 74 73 98.64 71 97.26
1 5 (71.42%)
2 71 1.91 10 (27.02) 8 (21.62) 14.08
8 (80%)
Gradea
No. of embryos 2-cell
3-cell
4-cell
>4-cell
Total
Home-made media A 8 B 8 C 1 D 3 Total 20
1 3 3 1 8
16 16 6 2 40
3 0 0 0 3
28 27 10 6 71
Commercial media A 8 B 6 C 3 D 4 Total 21
2 0 3 0 5
5 8 7 0 20
1 0 1 0 2
16 14 14 4 48
a
According to Steer et al. (19).
5. Discussion Over the past decade the number of reported live births resulting from the transfer of vitrified-warmed human embryos has rapidly increased (20,21). To solve the problem of multiple pregnancies during the IVF and embryo transfer procedure, excess embryos could be cryopreserved for embryo transfer in the future. Furthermore, controlled ovarian hyperstimulation (COH) affects adversely implantation following IVF–embryo transfer (ET) (22). The endometrial development in frozenthawed cycles can be controlled more precisely than in the cycles of COH with gonadotropins (23). This results in an increased need for both oocytes and zygotes’ cryopreservation (24). Most of the successful and common vitrification methods published contain vitrification solution that includes EG with DMSO in combination at a concentration of up to 15% for each cryoprotectant (12,25,26). CM (RapidVitä Cleave) includes EG with PrOH and HM includes DMSO, EG as a cryoprotectant. Both media worked well with 2PN stage human zygotes when performed in Vitri 2ä Cleave for 2 min followed by in Vitri 3ä Cleave for 30 s at 37 °C, in protocol II following the established procedure of 8 min exposure in an equilibration solution and 1 min in vitrification solution at room temperature. Faster equilibration may allow shorter exposure to the first cryoprotectant solution (Vitri 2ä Cleave) and thus also has less risk for any potential toxicity on membranes or intracellular components. On the other hand, a shorter exposure does not allow shrinkage of the zygotes. There was one monozygote twin in protocol I. Monozygotic twin pregnancies have been reported to occur at a significantly higher rate following ART procedures compared with the natural incidence (27,28). A cryoprotectant mixture with high glass-forming ability, low toxicity and low viscosity is the elusive goal of vitrification research. Recently, it has been shown that cryoprotectants can permeate the cell membrane via aquaporins (29,30). EG has been widely used during vitrification for both human oocytes and embryos due to its low toxicity and high permeability (31–33). It diffuses into and leaves the embryos very rapidly owing to its low molecular weight, thus embryos may undergo less osmotic stress during vitrification and warming. High con-
192 centrations of cryoprotectants together with rapid cooling rates are necessary for a successful vitrification method. However, the higher concentration of cryoprotectants needed for this method may cause cryoprotectant toxicity. The introduction of cryoprotectants with higher membrane permeability and lower toxicity, and possible use of combinations of nonpermeable cryoprotectants, overcome cytotoxicity (34). Various substances including polymers with low toxicity have been suggested for this purpose. However, the traditionally used sucrose or trehalose seem to be most appropriate as well as other molecules with increasing viscosity such as ficoll. They also act as an osmotic buffer, preventing ‘osmotic shock’ following dilution of the cryoprotectant after warming procedure. Nonetheless, non-penetrating cryoprotectants can assist vitrification because most nucleators are extracellular and dehydration allows for intracellular vitrification by bound water. Extracellular vitrification which involves sugar prevents cell membranes from coming in contact and fusing (35). Cell membranes are commonly believed to be the part of cells most vulnerable to freezing damage. Recently, Kartberg et al. showed that differences occur in membrane integrity when comparing EG–PrOH with EG–PrOH–DMSO. DMSO containing vitrification solutions did not lead to cell membrane damage and death as quickly as the DMSO free vitrification solutions. However, differences became apparent only after 10 min or longer exposure to the vitrification solution (36). The results of vitrification were significantly better compared to slow freezing by using the vitrification solution that contained PrOH (37). Vitrification either at blastocyst stage or cleavage and PN stage of embryo seems to be favorable and efficient in view of an increased outcome such as survival and pregnancy rates (15,38,39). On the other hand, more advanced pregnancy rates have been reported by vitrification at blastocyst stage. Since morphology alone of a vitrified and thawed embryo is not enough to assess viability, the possibility of culturing for a few more days before transfer can ensure that a viable embryo will be used for transfer. This is also advantageous as it was remarked that embryo transfer at day 5, in comparison to day 2 or 3, gives an increased pregnancy and implantation rate (40,41). Vitrification at early stages is became a more popular alternative to the vitrification at 2PN-stage and will serve quite well in countries where further culturing and embryo selection is not allowed. Vitrification is a very simple, cost-effective process and the skills needed to perform it can be acquired in a short time through training on materials (42,43). Today it seems that vitrification is rapidly spreading all over the world and will be the future of cryopreservation. Previous studies indicated that the homemade vitrification solution which has been used routinely in the authors’ center for more than 5 years for 2PN stage zygotes which were used according to German embryo protection law gave good results (13). In conclusion, the two standard vitrification protocols, commercial kit and home-made solution, did not differ in 2PN zygotes on the parameters that we compared. Both vitrification solutions provided high survival rate and good outcomes. With the healthy live births following vitrification, both methods can, therefore, be adopted, without any doubt, as a safe procedure for human 2PN stage zygotes. To our knowledge, this is the first comparison between different media in a vitrification procedure.
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