- Email: [email protected]
How does closed system vitrification of human oocytes affect the clinical outcome? A prospective, observational, cohort, noninferiority trial in an oocyte donation program
ORIGINAL ARTICLE: ASSISTED REPRODUCTION
How does closed system vitrification of human oocytes affect the clinical outcome? A prospective, observational, cohort, noninferiority trial in an oocyte donation program Achilleas Papatheodorou, Ph.D.,a,b Pierre Vanderzwalmen, Ph.D.,c,d Yannis Panagiotidis, Ph.D.,a Stamatios Petousis, M.D., Ph.D.,a,e Giuseppe Gullo, M.D.,f Evangelia Kasapi,a Maria Goudakou, Ph.D.,a Nikos Prapas, M.D., Ph.D.,a Kostas Zikopoulos, M.D., Ph.D.,b Ioannis Georgiou, Ph.D.,b and Yannis Prapas, M.D., Ph.D.a a IAKENTRO Advanced Medical Center, Thessaloniki, Greece; b Center for Reproductive Medicine, Obstetrics and Gynecology, Ioannina University, Ioannina, Greece; c IVF Centers Prof Zech, Bregenz, Austria; d Chirec, Brussels, Belgium; e 3rd Department of Obstetrics and Gynaecology, Aristotle University, Thessaloniki, Greece; and f Department of Obstetrics and Gynaecology, University of Messina, Messina, Italy
Objective: To evaluate whether is possible to vitrify oocytes in an aseptic (hermetically closed) fashion and maintain clinical results comparable with those of fresh oocytes. Design: Prospective, observational, cohort, noninferiority trial. Setting: Private in vitro fertilization center. Patient(s): One hundred eighty-four recipients of donated vitrified oocytes. Intervention(s): Closed system vitrification. Main Outcome Measure(s): Pregnancy rate per cycle and clinical pregnancy rate per cycle. Result(s): No statistically significant differences were observed between two groups regarding the pregnancy rate per cycle (63.1% vs. 60.9%) or the clinical pregnancy rate per cycle (55.4% vs. 58.7%). Biochemical pregnancy rate was statistically significantly higher in the fresh group (7.6% vs. 2.2%). The mean number of embryos transferred was similar (2.0 0.0 vs. 1.97 0.3). Concerning embryologic data, there were no statistically significant differences regarding the fertilization, cleavage, top quality day-3 embryo, or blastocyst rates, whereas the top quality blastocyst rate on day 5 was statistically significantly higher in the fresh oocyte group (31.7% vs. 26.1%). Conclusion(s): Aseptically (in a closed system) vitrified oocytes show similar clinical efficiency compared with their sibling fresh oocytes. (Fertil SterilÒ 2016;-:-–-. Ó2016 by American Society for Reproductive Medicine.) Key Words: Aseptic technique, closed system vitrification, egg donor bank, oocyte donation, oocyte vitrification Discuss: You can discuss this article with its authors and with other ASRM members at https://www.fertstertdialog.com/users/16110fertility-and-sterility/posts/11072-how-does-closed-system-vitrification-of-human-oocytes-affect-the-clinical-outcome-a-prospectiveobservational-cohort-noninferiority-trial-in-an-oocyte-donation-program
F
or over 15 years vitrification as a cryopreservation technique has been successfully applied to
human embryos. From the moment that vitrification was proven to be a promising alternative to cryopreserve
Received February 5, 2016; revised June 17, 2016; accepted July 11, 2016. A.P. has nothing to disclose. P.V. has nothing to disclose. Y.P. has nothing to disclose. S.P. has nothing to disclose. G.G. has nothing to disclose. E.K. has nothing to disclose. M.G. has nothing to disclose. N.P. has nothing to disclose. K.Z. has nothing to disclose. I.G. has nothing to disclose. Y.P. has nothing to disclose. Reprint requests: Achilleas Papatheodorou, Ph.D., IAKENTRO Fertility Center, Agiou Vasileiou 4, 54250, Thessaloniki, Greece (E-mail: [email protected]). Fertility and Sterility® Vol. -, No. -, - 2016 0015-0282/$36.00 Copyright ©2016 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2016.07.1066 VOL. - NO. - / - 2016
oocytes, the popularity of the technique in the field of assisted reproduction technology (ART) has grown (1, 2). Many published studies have reported the superiority of vitrification over the slow freezing technique for oocyte cryopreservation, and the success rates using vitrified oocytes have been similar to those obtained with fresh oocytes (2–13). The birth of healthy infants resulting from vitrified oocytes (14–17) has established vitrification as 1
ORIGINAL ARTICLE: ASSISTED REPRODUCTION the gold standard technique for oocyte cryopreservation and has widened the indications for use of this technique. In the past, regardless the disappointing results, oocyte cryopreservation was the only option in specific circumstances, such as for women at risk of losing their ovarian reserve (from cancer, immunologic or genetic disorders, or aggressive medical treatments), for overcoming legal, ethical, and religious restrictions, or for addressing logistic situations such as an absent semen sample on the day of oocyte pickup. At present, due to the promising results and the safety of the technique, as demonstrated in healthy infants, vitrification is no longer considered an experimental method (18). With the introduction of efficient, harmless, and safe vitrification techniques, the indications for vitrifying oocytes could be widened and proposed to women who are seeking fertility preservation for social reasons or used to promote the development of egg donor banks. Noticeable is the fact that a longer period of storage, ranging from months to several years, will be needed if the demand for egg freezing increases in the future (19). In such prolonged storage conditions, safety is another key issue because the biological sample must be aseptically isolated and not in direct contact with liquid nitrogen (20). To guarantee aseptic vitrification and storage conditions, closed carrier systems have been introduced (21, 22). At present, few data using closed devices that ensure aseptic cooling and storage have been reported in cases of oocytes cryopreservation. Recent reviews (1, 23) have noted the obvious preference of scientists for using open systems for oocyte vitrification. This reluctance to vitrify using closed devices derives from a common belief (2, 23–25) that reduced cooling rates, such as those produced in closed systems due to thermo-isolation, could be harmful or lethal to the cells, increasing the probability of ice crystal formation during the cooling process (26). However, it has been reported that reduced cooling rates do not compromise survival rates in aseptically vitrified oocytes (27–30), zygotes (30, 31), or blastocysts (21, 32, 33) if very high warming rates are applied. Such statements are reinforced by recent studies of Mazur et al. (34–37), who have shown that the primary cause for cell injury or cell death during vitrification procedure is not the ice crystal formation during cooling but the recrystallization during warming (devitrification). According to their studies, warming rates are as important as cooling rates; in fact, warming rates should be higher than cooling rates for a successful vitrification and warming cycle. Therefore, a closed system could be as sufficient as an open one so long as we keep the warming rates high. So far, a few studies have described the competence of closed systems for oocyte vitrification (5, 27, 32, 38), but their clinical data are not enough to support the efficiency of these devices. Unlike the open systems, there are no prospective studies comparing fresh embryo transfers (ET) versus ET after oocyte vitrification in hermetically closed devices. To evaluate the efficiency of hermetically closed devices in oocyte vitrification we compared the clinical 2
outcome after ET derived from sibling oocytes to recipients being synchronized (fresh oocytes) or not (aseptically vitrified oocytes) with their donor in our oocyte donation program.
MATERIALS AND METHODS A prospective, observational, cohort study was performed at IAKENTRO Fertility Center from recruitment start date on January 19, 2014, to the completion date on December 15, 2014. All procedures were performed at the same laboratory. This clinical trial was a part of a doctoral study at the Medical School of University of Ioannina, Greece. The study was approved by the institutional review board of the Ioannina Medical School (Ref: 808a/8-3-2011). This trial was registered in ISRCTN registry (identification number ISRCTN56275481) and was approved by the IAKENTRO review board (reference number 1/2014, 19/1/2014). Informed consent was obtained from all women participating.
Noninferiority Test, Sample Size, and Study Design Based on a positive hCG/transfer baseline rate of 61% among controls and 59% for study subjects, a sample size of 92 transfers per arm would be required to be able to reject the null hypothesis that the one-sided 95% confidence interval (or equivalently a 90% two-sided confidence interval) will exclude a difference in favor of the standard group of more than 20% with a statistical significance level of 5% and a power (1-b) of 80%. Power calculation was performed with Sealed Envelope Ltd 2012 (39). Ninety-two oocyte donors participated in our study. A single stimulation cycle was included for each donor. Pairs of recipients, sharing sibling oocytes from the same donor, were included in the study. Each pair consisted of a recipient for whom fresh oocytes were used for their donation cycle and another recipient for whom the oocytes were vitrified and used after a short period of time. A single donation cycle was included for each recipient. One hundred and eightyfour couples who received sibling oocytes donated from the same donor were allocated to receive fresh (92 couples) or vitrified sibling oocytes (92 cases). Biological and clinical parameters were evaluated. Pregnancy rates were a secondary tracked outcome; the intervention did not depend on it, nor did it affect the execution of the study in any way. The current study contains a randomization procedure. During the donor's oocyte pickup, two separate dishes for oocyte collection were used. The retrieved cumulus oocyte complexes were randomly and equally assigned into the two dishes during oocytes retrieval. Odd numbered oocytes were allocated to the fresh group (dish 1: group 1), and even numbered oocytes were allocated to the closed vitrified group (dish 2: group 2). This allocation method could potentially have the drawback of recruiting a higher number of oocytes to group 1 as in all odd-numbered cases group 1 would have more oocytes enrolled. However, according to our experience, the larger follicles are first retrieved in each procedure are more likely to contain higher quality (mature) oocytes, so it is likely that the last-retrieved odd oocytes would be of poor VOL. - NO. - / - 2016
Fertility and Sterility® quality (immature) and would not finally result in a significantly different number of metaphase 2 (MII) oocytes in each group. Furthermore, it would demand an additional randomization process to decide which cases' oocyte allocation would begin from group 1 and from group 2. Therefore, to avoid complexity of procedure and because of the profound lack of clinical impact, it was initially decided to recruit oocytes in the same manner for all cases. Fresh group oocytes (group 1) were fertilized the same day, and vitrified group oocytes (group 2) were stored in liquid nitrogen tanks. The warming and the fertilization of group 2 oocytes were performed when the recipient to whom those oocytes had been allocated had her endometrium properly prepared.
Inclusion Criteria A detailed medical history of the donors was taken. The oocyte donors were %32 years old, had body mass index <30 kg/m2, regular menstrual cycles of 25–35 days, two normal ovaries based on transvaginal scan findings, no polycystic ovary syndrome, no known endometriosis, and no gynecologic or medical disorders. They all had agreed to donate their oocytes for treatment anonymously and altruistically. Oocyte donors were of known fertility and good ovarian response. Blood samples were collected for karyotyping and screening for previous viral infections (hepatitis B and C, human immunodeficiency virus, syphilis), thalassemia, and cystic fibrosis. Our study included patients with a minimal of eight mature oocytes, which is a basic requirement offered to recipient couples in our donation program. Consequently, after the denudation of retrieved oocytes, if a recipient wound up with %7 mature oocytes, these cycles were excluded from our analysis because one of the two recipient cycles had to be canceled. A total of 184 recipients, matched with their donors (n ¼ 92), were included in the study. All recipients were <50 years old with no history of endometriosis, and this was their first oocyte donation cycle. The recipients and their partners underwent blood screening similar to the donors, and a hysterosalpingogram and a diagnostic hysteroscopy eliminated cases presenting hydrosalpinx or intrauterine-related pathology. The recipients went through a mock transfer in a cycle before their donation cycle; if any difficulty was encountered, a cervical dilatation was performed (40). In both groups, the recipient couples had no form of severe male infertility indications. All the enrolled cases had normal values for concentration, motility, and morphology. The World Health Organization criteria (41) were used to evaluate the sperm quality. For each oocyte donation cycle, a recipient was allocated to receive the fresh and another to receive the vitrified oocytes, with an identical procedure: our center has a list of patients available for fresh oocytes and another list of patients available only for vitrified oocytes. Among all patients who had the same blood type and similar morphologic characteristics with the oocyte donor, there was one recipient from each list allocated by computerized random select software for each oocyte donation cycle. VOL. - NO. - / - 2016
Stimulation Protocol and Recipient Preparation The ovarian stimulation of the donors was performed with a fixed day-6 gonadotropin-releasing hormone (GnRH) antagonist protocol (42). Recipients in the fresh group underwent endometrial preparation as previously described elsewhere (43). Women with ovarian function were first downregulated in the luteal phase with a single-dose of GnRHagonist depot (Arvekap, 3.75 mg) beginning on day 21 of the previous cycle. One day after the announcement of the donor's period onset, the recipients were instructed to begin administering estradiol valerate (Cyclacur or Progynova) at 2 mg/d for the first 4 days, 4 mg/d for days 5–8, and 6 mg/d until the pregnancy test. The afternoon of the oocyte donation pickup, the recipients were administered 200 mg of progesterone (Utrogestan) intravaginally; this continued as 200 mg, three times a day, until a fetal heartbeat was observed by ultrasound. The recipients without menstruation followed the same protocol without the GnRH agonist. Endometrial development was evaluated by ultrasound scan; it was considered mature when the endometrial thickness was >9 mm. Recipients in the vitrification group followed the same treatment as fresh group recipients. They began with a GnRH agonist on the day 21 of the previous cycle. After confirming down-regulation by measuring serum estradiol and progesterone concentrations, we administered estrogen as mentioned previously. Endometrial development was evaluated by ultrasound scan; it was considered mature when the endometrial thickness was >9 mm. If the endometrium was <9 mm, the transfer cycle was canceled. The embryos of the fresh oocyte group were vitrified on day 5, and the transfer was postponed for a future cycle. These cycles and their sibling cases were not included in our cohort. In vitrified oocyte group, when the endometrium was <9 mm, the oocytes were not warmed, and the cycle was postponed for a future date (with new endometrial preparation).
Laboratory Procedures The oocytes retrieved from each donor were equilibrated in Single Step Medium (SSM; Irvine Scientific) for 2 hours and then enzymatically denuded (hyaluronidase solution; Irvine Scientific). Only MII oocytes were included in the study groups. Fresh group (group 1) oocytes were injected 1 hour after denudation. Closed vitrification group (group 2) oocytes were vitrified a half-hour to 1 hour after denudation. After their warming, oocytes were cultured in SSM for 3 hours before they were injected. Intracytoplasmic sperm injection (ICSI) was performed in all cases. Fertilization was assessed 16 to 20 hours after ICSI by visualization of the two pronuclei. Embryos were cultured in 30-mL droplets of SSM, overlaid with mineral oil (FertiCult Mineral Oil - FERTIPRO) in a MINC benchtop incubator (Cook Medical) with provided triple gas of 6% CO2, 5% O2, and 89% N2. Embryo quality was assessed on days 3 and 5. For day-3 embryos, the number of cells, the appearance of blastomeres, and the presence of cytoplasm defects or fragmentation were evaluated (44). According to this system, top quality cleaved embryos were considered those with 8 to 12 symmetric blastomeres with absence of cytoplasm defects and without or with negligible fragmentation. 3
ORIGINAL ARTICLE: ASSISTED REPRODUCTION For the evaluation of day-5 embryos we used the Gardner and Schoolcraft criteria (45) in which thin zona pellucida, smooth trophectoderm, equality and close adhesion of blastomeres, clearly visible blastocyst cavity, and well-developed inner cell mass with many closely aggregated cells are the most important parameters correlating to top blastocyst quality. We performed ET performed on day 5 under ultrasound guidance as previously described elsewhere (46). The remaining embryos were vitrified on day 5 using a closed vitrification system.
Oocyte Vitrification in a Closed System Oocytes were vitrified by using the Vitrisafe carrier (21) (VitriMed) at a maximum of 3 hours after oocyte pickup. Denuded oocytes were exposed to four equilibration solutions with 1.25%, 2.5%, 5%, and 10% concentrations of dimethyl sulfoxide and ethylene glycol (FertiVit Cooling Kit - FERTIPRO) for 3, 3, 3, and 5:30 minutes, respectively, and one vitrification solution with 20% dimethyl sulfoxide and 20% ethylene glycol plus 0.75 mol/L sucrose and 10 mg/L Ficoll (FertiVit Warming Kit - FERTIPRO) for 1 minute. After exposure to the cryoprotectants, by means of a single drop the oocytes were initially transferred on the Vitrisafe, and then the carrier was inserted in the protective straw, which was thermosealed and plunged into a Dewar with liquid nitrogen. All procedures were performed at room temperature (25 C). Warming was performed in five steps as described elsewhere (28). Briefly, the oocytes were exposed to gradually decreased concentrations—1 M, 0.75 M, 0.5 M, 0.25 M, and 0.125 M—of sucrose for 1, 1, 2, 2, and 2 minutes, respectively (Fertipro). The rehydration of the oocytes was performed at room temperature (25 C) with an exception of the first step, in which the Vitrisafe was immersed in a warm solution (37 C) of 1 M sucrose for 1 minutes. To recover, the oocytes were placed into culture medium (SSM) and incubated for 3 hours at 37 C (6% CO2 and 5% O2) before they were injected. Degenerated oocytes were removed from the cohort.
Outcome Measures The number of surviving oocytes, fertilized oocytes, cleavage embryos, top quality cleavage embryos, blastocysts, top quality blastocysts, and embryos transferred were analyzed. Oocyte survival rate was defined as the number of oocytes that survived out of the total number of oocytes warmed. We applied ICSI with all surviving oocytes. Fertilization rate was defined as the number of fertilized oocytes out of the number of oocytes that survived. Cleavage rate, top cleavage rate, blastocyst rate, and top quality blastocyst rate were defined as the number of cleavage embryos, top quality cleavage embryos, blastocysts, and top quality blastocysts, respectively, out of the total number of mature (MII) oocytes. Pregnancy was confirmed by the rise of serum b-human chorionic gonadotropin (hCG) concentration 14 days after ET. Clinical pregnancy was defined by the appearance of a gestational sac and a fetal heartbeat at 8 to 10 weeks of gestation. 4
Ongoing pregnancy rate was considered the number of pregnancies with fetuses displaying heart activity beyond 12 weeks of gestation per cycle. The miscarriage rate was considered the loss of a clinical pregnancy per cycle before 20 completed weeks of gestation. Implantation rate was defined as the number of gestational sacs per transferred embryos. As biochemical pregnancy was considered the initial rise and the immediate fall of hCG in serum level verified by the absence of a gestational sac under the ultrasound. As a private in vitro fertilization (IVF) center and not a reference center or a public hospital, we did not monitor the couples in the study by ourselves. Live births were reported to our clinic after communication with the couple or their gynecologist. Babies who had no reports of anomalies were considered as healthy.
Primary and Secondary Outcomes The primary end point of the study was the positive hCG rate per cycle. Secondary outcomes were clinical pregnancy rate per cycle, ongoing pregnancy rate, biochemical pregnancy rate, miscarriage rate, twinning rate, and live-birth rate. Secondary end points were also the oocyte survival rate, the fertilization rate, the cleavage and top cleavage rates, and the blastocyst and top blastocyst rates.
Statistical Analysis Continuous data are expressed as mean standard deviation, and categorical data as percentages (%). Normality was assessed with the Kolmogorov-Smirnov test. An independent sample t test was used for parametric and Mann-Whitney test for nonparametric numeric variables. Categorical data were analyzed with chi-square analysis. The statistical analyses were performed using the Statistical Package for Social Sciences, version 17.0 (IBM/SPSS Inc.). The non-inferiority test was performed by using the power calculator of sealed envelope at www.sealedenvelope.com.
RESULTS Data from 184 recipients were prospectively collected from January 2014 to December 2014. No cases were included in the study who finally dropped out due to survival, fertilization, or blastocyst formation failures. The epidemiologic characteristics of the included cases were similar between the two groups (Table 1). A total of 2,325 oocytes were retrieved: 1,175 in group 1, and 1,150 in group 2. Of these, 1,966 of were MII: 982 in the fresh group and 984 in the closed vitrification group (Table 2). There was no difference in the number of the allocated oocytes between the two groups (10.7 2.1 vs. 10.7 2.0, P¼ .94), thus confirming the initial hypothesis that our oocyte allocation method would not result in a statistically significantly different number of MII oocytes. The survival rate of the oocytes from group 2 was 92.7% (912 of 984 oocytes). No statistically significant difference was observed regarding the fertilization rate, cleavage rate, top quality embryos on day 3, or blastocyst rate between the two groups. The top quality blastocyst rate on day 5 was statistically significantly VOL. - NO. - / - 2016
Fertility and Sterility®
TABLE 1 Baseline characteristics of donors, recipients, and the recipients' male partners. Characteristics Donor Age (y) BMI (kg/m2) Stimulation (d, range) Total rFSH (IU) E2 (pg/mL) Recipient Age (y) Endometrium thickness BMI (kg/m2) Endometrial preparation (d) Male partner Age (y) Sperm concentration (millions/mL) Motility Forward motility Morphology
Donor
Group 1 fresh oocytes (n [ 92)
Group 2 vitrified oocytes (n [ 92)
P value
43.2 3.4 10.9 1.1 23.8 3.5 16.9 1.1
43.5 3.4 10.7 0.9 23.6 3.1 16.8 0.5
.57 .31 .61 .42
43.62 5.6 49.2 22.6 67.87 11.8 56.1 13.1 16.52 4.6
42.92 5.7 46.6 22.4 67.5 11.8 56.6 14.3 16.43 6.4
.41 .42 .85 .84 .91
28 3.2 22.6 2.6 9.8 1.1 (8–12) 2,192 407 2,230 640
Note: Values are mean standard deviation. BMI ¼ body mass index; E2 ¼ estradiol; rFSH ¼ recombinant follicle-stimulating hormone. Papatheodorou. Comparison of fresh and vitrified oocytes. Fertil Steril 2016.
higher in the fresh oocyte group (31.7% vs. 26.1%, P¼ .003). Embryologic parameters are presented in Table 2. Ninety-two transfers were performed in each group. The mean number of embryos transferred was similar (2.0 0.0 vs. 1.97 0.3, P¼ .25). No statistically significant difference was observed between two groups regarding pregnancy (bhCG positive) rate per cycle (63.1% vs. 60.9%, P¼ .76) or clinical pregnancy rate per cycle (55.4% vs. 58.7%, P¼ .66). No statistically significant differences were observed regarding all the other outcomes between the two groups. Regarding live births, 66 healthy babies were born from the fresh group and 62 from the vitrification group. No adverse outcome was reported by the parents. The primary
and secondary clinical outcomes of the present study are presented in Table 3. We have calculated that the collected oocytes to baby ratio was 5.6% and the MII oocytes to baby ratio was 6.8% in the fresh group. In the vitrification group the ratios were 5.3% and 6.3%, respectively (Table 4). The MII oocytes needed to achieve a pregnancy were 14.8 in fresh group and 15.8 in the vitrification group (Table 4). The power analysis according to observed rates (63.1% for controls and 60.9% for patients) indicated that, with 92 women in each group, our study had a 80.5% power to reject the null hypothesis that one-sided 95% confidence interval (or equivalently a 90% two-sided confidence interval) will
TABLE 2 Comparison of the biological parameters of group 1 (fresh) and group 2 (vitrified). Parameters Retrieved oocytes MII oocytes Vitrified oocytes Survived oocytes Survival rate, % Fertilized oocytes Fertilization rate, % Cleaved embryos (d 3) Cleavage rate, % Top quality cleaved embryos Top quality cleavage rate, % Blastocysts Blastocyst rate, % Top quality blastocyst Top quality blastocyst rate, % Embryos transferred Vitrified embryos
Group 1 fresh oocytes (n [ 92)
Group 2 vitrified oocytes (n [ 92)
P value
12.7 2.4 (1,175) 10.7 2.1 (982) – – – 8.6 1.8 (796) 81.1% (796/982) 8.0 1.6 (739) 75.2% (739/982) 4.3 2.0 (403) 41.1% (403/982) 5.2 1.7 (484) 49.3% (484/982) 3.4 2.0 (312) 31.7% (312/982) 2.0 0.0 (184) 2.76 1.5 (254)
12.5 2.1 (1,150) 10.7 2.0 (984) 10.7 2.0 (984) 9.9 2.1 (912) 92.7% (912/984) 8.1 1.7 (744) 81.6% (744/912) 7.5 2.0 (692) 75.8% (692/912) 4.1 2.1 (382) 41.8% (382/912) 4.8 2.1 (440) 48.2% (440/912) 2.6 1.6 (238) 26.1% (238/912) 1.96 0.4 (180) 2.41 1.8 (222)
.86 .94 – – – .23 .77 .29 .90 .37 .80 .10 .51 .003 .003 .25 .16
Note: Values are mean standard deviation (number) unless otherwise indicated. Papatheodorou. Comparison of fresh and vitrified oocytes. Fertil Steril 2016.
VOL. - NO. - / - 2016
5
ORIGINAL ARTICLE: ASSISTED REPRODUCTION
TABLE 3 Number of embryos transferred and clinical parameters per cycle.
Parameters Positive b-hCG Clinical pregnancy rate Ongoing pregnancy rate Implantation rate Biochemical pregnancy rate Miscarriage rate Delivery rate Twin pregnancy rate Live births
Group 1 fresh oocytes (n [ 92)
Group 2 vitrified oocytes (n [ 92)
P value
63.1% (58/92) 55.4% (51/92)
60.9% (56/92) 58.7% (54/92)
.76 .66
51.1% (47/92)
52.2% (48/92)
.88
41.8% (77/184) 7.6% (7/92)
38.9% (70/180) 2.2% (2/92)
.57 .05
4.3% (4/92) 51.1% (47/92) 40.4% (19/47) 66
6.5% (6/92) 52.1% (48/92) 29.2% (14/48) 62
.52 .88 .25 .23
Note: b-hCG ¼ b-human chorionic gonadotropin. Papatheodorou. Comparison of fresh and vitrified oocytes. Fertil Steril 2016.
exclude a difference in favor of the standard group of more than 20%.
DISCUSSION Our prospective comparative study shows the parity of fresh and sibling vitrified oocytes. No differences were observed regarding the pregnancy rate, clinical pregnancy rate, ongoing pregnancy rate, or live-birth rate between the fresh and vitrified oocytes. Additionally, it has been shown that fresh and sibling vitrified oocytes have similar developmental potential and implantation capacity. The only statistically significant differences we obtained were a higher top quality blastocyst rate on day 5 in favor of the fresh group and a lower biochemical pregnancy rate in favor of the vitrification group. In this study, to eliminate confounding effects related to female age and ovarian stimulation, a population of oocyte donors and their sibling recipients who shared the oocytes of the same donor were studied. Furthermore the design of the current study shows several strengths. To our knowledge, this work is the only prospective study with the following characteristics: [1] prospective study with randomization, [2] sibling donated oocytes, [3] one fresh group and one vitri-
TABLE 4 Oocyte to baby ratio and oocytes used to achieve a pregnancy.
Parameter Collected oocytes to baby ratio Collected oocytes used to achieve a live birth rate MII oocytes to baby ratio MII oocytes used to achieve a live birth rate
Group 1 fresh oocytes (n [ 92)
Group 2 vitrified oocytes (n [ 92)
5.6% 17.8
5.3% 18.5
6.8% 14.8
6.3% 15.8
Note: MII ¼ metaphase 2. Papatheodorou. Comparison of fresh and vitrified oocytes. Fertil Steril 2016.
6
fication group, [4] vitrification of oocytes performed in a closed system (aseptic conditions), and [5] blastocyst culture and transfer performed in both groups. Because of these characteristics, we consider our findings of high importance. The ultimate proof of vitrified oocyte competency is to achieve comparable live-birth rates to fresh oocytes. Using the open vitrification technique in an oocyte donation program Cobo et al., obtained similar outcomes between fresh and vitrified oocytes (11). Later, Trokoudes et al. (47) reported comparable results obtained with vitrified oocytes using the open technique. Our study in an oocyte donation program has shown that the same results can be achieved with a closed vitrification device because the implantation, pregnancy, clinical pregnancy, and ongoing pregnancy rates were no different between the vitrified and fresh sibling oocytes. Cobo et al. (19) have reported on the oocyte to baby ratio (6.5%: 6.5 babies born for every 100 oocytes used) and the number of oocytes needed (15.4) to achieve a baby. These results are taken from a large database of 32,460 heterologous and 1,513 autologous oocytes. In our study the collected oocytes to baby ratio was 5.6% and the MII oocytes to baby ratio was 6.8% in the fresh group; in the vitrification group the ratios were 5.3% and 6.3%, respectively (Table 4). The MII oocytes needed to achieve a pregnancy were 14.8 in fresh group and 15.8 in the vitrification group (Table 4). In both studies, we see that a relatively big number of MII oocytes (around 15) are needed to achieve a live birth. In fact, Cobo et al. (19) report that the more oocytes used, the higher the cumulative pregnancy rate. When five oocytes are used the rate is low, and it statistically significantly increases as the number of consumed oocytes doubles. It is very important to notice that these numbers are referring to oocytes donated from young healthy donors. We must be very careful to not extrapolate the above ratios to women of different age groups or different fertility statuses. The oocyte to baby ratio would be completely different for these women, so we must be careful when we advise them to cryopreserve their oocytes. This is something that is mentioned by Cobo et al. (19), who calculated that women over 40 years old should consume 55.5 oocytes to achieve the live-birth rate. Looking at the vitrification group we observed a survival rate of 92.7% after warming. This result is similar to those reported in open vitrification system studies (11), and the average reported in a recent systematic review and metaanalysis (1). The fertilization, cleavage, top cleavage, and blastocyst rates were similar between the vitrified oocytes and fresh groups and in accordance with recent studies that have used vitrified donor oocytes (1). The scope of our study was not to compare closed and open vitrification devices, but comparing the efficiency of the devices was inevitable. The parity of closed versus open vitrification devices has been well known to our team since 2009 (21, 48) and is well established by randomized, controlled trials in embryos (32) and oocytes (33). Nonetheless, scientists have an obvious preference for open system devices (1, 23) due to a common belief that high cooling rates, accomplished by direct contact of the sample with liquid nitrogen, are the key point to successful oocyte vitrification. This belief was challenged by Seki and Mazur VOL. - NO. - / - 2016
Fertility and Sterility® (34–36) who proved that warming rates are of equal or higher importance as the cooling rates. They showed that once a certain high warming rate is achieved, the initial cooling rate is of less importance; if you warm fast enough, the cells will survive. Our study has confirmed their findings because the closed devices used for the vitrification could maintain the clinical efficiency of the vitrified oocytes. It was shown that although the cooling rates of closed system devices are lower than the equivalent of open system, they are still high enough and in combination with a very rapid warming procedure were able to obtain excellent survival rates and demonstrate the competency of the vitrified oocytes. The development of a reliable and safe aseptic (closed) vitrification protocol is of high importance for human tissue cryopreservation, especially after the Bielanski's reports on possibility of cross-contamination under liquid nitrogen that have raised skepticism on the use of open vitrification protocols (49, 50). The guidelines of European Parliament (European Parliament and the Council of the European Union, 2004, 2006) has impelled scientist to look for solutions that would maintain vitrification in an aseptic status (51, 52)—for instance, liquid nitrogen sterilized by filtration (53) or UV irradiation (54) or the storage of samples in vapors of nitrogen (25, 55). The greatest question has been how to solve the problem using the open system devices. The closed systems were examined and considered as potentially harmful for the cells due to the lower cooling rates. The results of our study have shown that closed vitrification systems are sufficient and can be used as devices to store biological samples in the safest way. At present, with an increase of social egg freezing and oocyte donation banking, we can expect a longer storage period for these oocytes, ranging from some months to several years. In such prolonged conditions our main concern is to store biological material in the safest way. A closed system device could guarantee the appropriate isolation from any detrimental factors such as the toxic low-molecular-weight compounds found in liquid nitrogen (56) whose long-term actions are not known. Our prospective comparative study sends an important message: vitrified oocytes have similar clinical efficiency to fresh oocytes. Additionally, their efficiency can be maintained even if we use a closed system for vitrification, which leaves behind any concerns regarding the biosafety of vitrified human tissue.
REFERENCES 1.
2.
3.
4.
Potdar N, Gelbaya TA, Nardo LG. Oocyte vitrification in the 21st century and post-warming fertility outcomes: a systematic review and meta-analysis. Reprod Biomed Online 2014;29:159–76. Kuwayama M, Vajta G, Kato O, Leibo SP. Highly efficient vitrification method for cryopreservation of human oocytes. Reprod Biomed Online 2005;11:300–8. Cao Y-X, Xing Q, Li L, Cong L, Zhang Z-G, Wei Z-L, et al. Comparison of survival and embryonic development in human oocytes cryopreserved by slowfreezing and vitrification. Fertil Steril 2009;92:1306–11. Rezazadeh Valojerdi M, Eftekhari-Yazdi P, Karimian L, Hassani F, Movaghar B. Vitrification versus slow freezing gives excellent survival, post
VOL. - NO. - / - 2016
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23. 24.
warming embryo morphology and pregnancy outcomes for human cleaved embryos. J Assist Reprod Genet 2009;26:347–54. Smith GD, Serafini PC, Fioravanti J, Yadid I, Coslovsky M, Hassun P, et al. Prospective randomized comparison of human oocyte cryopreservation with slow-rate freezing or vitrification. Fertil Steril 2010;94:2088–95. Ubaldi F, Anniballo R, Romano S, Baroni E, Albricci L, Colamaria S, et al. Cumulative ongoing pregnancy rate achieved with oocyte vitrification and cleavage stage transfer without embryo selection in a standard infertility program. Hum Reprod 2010;25:1199–205. Cobo A, Diaz C. Clinical application of oocyte vitrification: a systematic review and meta-analysis of randomized controlled trials. Fertil Steril 2011; 96:277–85. J, Boada M, Clua E, Rodríguez I, Martínez F, et al. How does Sole M, Santalo vitrification affect oocyte viability in oocyte donation cycles? A prospective study to compare outcomes achieved with fresh versus vitrified sibling oocytes. Hum Reprod 2013;28:2087–92. Levi Setti PE, Porcu E, Patrizio P, Vigiliano V, de Luca R, d'Aloja P, et al. Human oocyte cryopreservation with slow freezing versus vitrification. Results from the National Italian Registry data, 2007–2011. Fertil Steril 2014;102: 90–5.e2. Cobo A, Kuwayama M, Perez S, Ruiz A, Pellicer A, Remohí J. Comparison of concomitant outcome achieved with fresh and cryopreserved donor oocytes vitrified by the Cryotop method. Fertil Steril 2008;89:1657–64. Cobo A, Meseguer M, Remohí J, Pellicer A. Use of cryo-banked oocytes in an ovum donation programme: a prospective, randomized, controlled, clinical trial. Hum Reprod 2010;25:2239–46. Cobo A, Garrido N, Crespo J, Jose R, Pellicer A. Accumulation of oocytes: a new strategy for managing low-responder patients. Reprod Biomed Online 2012;24:424–32. Rienzi L, Cobo A, Paffoni A, Scarduelli C, Capalbo A, Vajta G, et al. Consistent and predictable delivery rates after oocyte vitrification: an observational longitudinal cohort multicentric study. Hum Reprod 2012;27: 1606–12. Chian R-C, Huang JYJ, Tan SL, Lucena E, Saa A, Rojas A, et al. Obstetric and perinatal outcome in 200 infants conceived from vitrified oocytes. Reprod Biomed Online 2008;16:608–10. Cobo A, Serra V, Garrido N, Olmo I, Pellicer A, Remohí J. Obstetric and perinatal outcome of babies born from vitrified oocytes. Fertil Steril 2014;102: 1006–15.e4. Noyes N, Porcu E, Borini A. Over 900 oocyte cryopreservation babies born with no apparent increase in congenital anomalies. Reprod Biomed Online 2009;18:769–76. Cai L-B, Qian X-Q, Wang W, Mao Y-D, Yan Z-J, Liu C-Z, et al. Oocyte vitrification technology has made egg-sharing donation easier in China. Reprod Biomed Online 2012;24:186–90. Practice Committees of American Society for Reproductive Medicine, Society for Assisted Reproductive Technology. Mature oocyte cryopreservation: a guideline. Fertil Steril 2013;99:37–43. Cobo A, Garrido N, Pellicer A, Remohí J. Six years' experience in ovum donation using vitrified oocytes: report of cumulative outcomes, impact of storage time, and development of a predictive model for oocyte survival rate. Fertil Steril 2015;104:1426–34.e8. Wirleitner B, Vanderzwalmen P, Bach M, Baramsai B, Neyer A, Schwerda D, et al. The time aspect in storing vitrified blastocysts: its impact on survival rate, implantation potential and babies born. Hum Reprod 2013;28:2950–7. Vanderzwalmen P, Ectors F, Grobet L, Prapas Y, Panagiotidis Y, Vanderzwalmen S, et al. Aseptic vitrification of blastocysts from infertile patients, egg donors and after IVM. Reprod Biomed Online 2009;19:700–7. Van Landuyt L, Stoop D, Verheyen G, Verpoest W, Camus M, Van de Velde H, et al. Outcome of closed blastocyst vitrification in relation to blastocyst quality: evaluation of 759 warming cycles in a single-embryo transfer policy. Hum Reprod 2011;26:527–34. Vajta G, Rienzi L, Ubaldi FM. Open versus closed systems for vitrification of human oocytes and embryos. Reprod Biomed Online 2015;30:325–33. Nagy ZP, Chang C-C, Shapiro DB, Bernal DP, Kort HI, Vajta G. The efficacy and safety of human oocyte vitrification. Semin Reprod Med 2009;27: 450–5.
7
ORIGINAL ARTICLE: ASSISTED REPRODUCTION 25.
26. 27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
8
Cobo A, Romero JL, P erez S, de los Santos MJ, Meseguer M, Remohí J. Storage of human oocytes in the vapor phase of nitrogen. Fertil Steril 2010;94: 1903–7. Rall WF, Fahy GM. Ice-free cryopreservation of mouse embryos at 196 degrees C by vitrification. Nature 1985;313:573–5. Stoop D, De Munck N, Jansen E, Platteau P, Van den Abbeel E, Verheyen G, et al. Clinical validation of a closed vitrification system in an oocyte-donation programme. Reprod Biomed Online 2012;24:180–5. Papatheodorou A, Vanderzwalmen P, Panagiotidis Y, Prapas N, Zikopoulos K, Georgiou I, et al. Open versus closed oocyte vitrification system: a prospective randomized sibling-oocyte study. Reprod Biomed Online 2013;26:595–602. De Munck N, Verheyen G, Van Landuyt L, Stoop D, Van de Velde H. Survival and post-warming in vitro competence of human oocytes after high security closed system vitrification. J Assist Reprod Genet 2013;30:361–9. Schiewe MC, Zozula S, Anderson RE, Fahy GM. Validation of microSecure vitrification (mS-VTF) for the effective cryopreservation of human embryos and oocytes. Cryobiology 2015;71:264–72. Vanderzwalmen P, Zech NH, Ectors F, Stecher A, Lejeune B, Vanderzwalmen S, et al. Blastocyst transfer after aseptic vitrification of zygotes: an approach to overcome an impaired uterine environment. Reprod Biomed Online 2012;25:591–9. Panagiotidis Y, Vanderzwalmen P, Prapas Y, Kasapi E, Goudakou M, Papatheodorou A, et al. Open versus closed vitrification of blastocysts from an oocyte-donation programme: a prospective randomized study. Reprod Biomed Online 2013;26:470–6. Papatheodorou A, Panagiotidis Y, Kasapi E, Goudakou M, Passadaki T, Prapas N, et al. Fresh vs aseptic vitrified sibling embryo transfers in oocyte donation program: a prospective, observational, comparative study. Hum Reprod 2013;28(Suppl 1):i62–4. Seki S, Mazur P. The dominance of warming rate over cooling rate in the survival of mouse oocytes subjected to a vitrification procedure. Cryobiology 2009;59:75–82. Mazur P, Seki S. Survival of mouse oocytes after being cooled in a vitrification solution to 196 C at 95 to 70,000 C/min and warmed at 610 to 118,000 C/min: a new paradigm for cryopreservation by vitrification. Cryobiology 2011;62:1–7. Seki S, Mazur P. Ultra-rapid warming yields high survival of mouse oocytes cooled to 196 c in dilutions of a standard vitrification solution. PLoS One 2012;7:e36058. Jin B, Mazur P. High survival of mouse oocytes/embryos after vitrification without permeating cryoprotectants followed by ultra-rapid warming with an IR laser pulse. Sci Rep 2015;5:9271. Paffoni A, Guarneri C, Ferrari S, Restelli L, Nicolosi AE, Scarduelli C, et al. Effects of two vitrification protocols on the developmental potential of human mature oocytes. Reprod Biomed Online 2011;22:292–8. Sealed Envelope. Power calculator for binary outcome non-inferiority trial. Available at: https://www.sealedenvelope.com/power/binary-noninferior/. Accessed April 22, 2016. Prapas N, Prapas Y, Panagiotidis Y, Prapa S, Vanderzwalmen P, Makedos G. Cervical dilatation has a positive impact on the outcome of IVF in randomly assigned cases having two previous difficult embryo transfers. Hum Reprod 2004;19:1791–5. World Health Organization. Laboratory manual for the examination and processing of human semen. 5th ed. Geneva: WHO; 2010.
42.
43.
44.
45.
46.
47.
48.
49. 50.
51.
52.
53.
54.
55.
56.
Prapas N, Prapas Y, Panagiotidis Y, Prapa S, Vanderzwalmen P, Schoysman R, et al. GnRH agonist versus GnRH antagonist in oocyte donation cycles: a prospective randomized study. Hum Reprod 2005; 20:1516–20. Prapas N, Tavaniotou A, Panagiotidis Y, Prapa S, Kasapi E, Goudakou M, et al. GnRH antagonists and endometrial receptivity in oocyte recipients: a prospective randomized trial. Reprod Biomed Online 2009;18:276–81. Veeck LL. An atlas of human gametes and conceptuses: an illustrated reference for assisted reproductive technology. New York: Taylor & Francis; 1999. Gardner D, Schoolcraft W. In vitro culture of human blastocyst. In: Towards reproductive certainty: infertility and genetics beyond. Carnforth, United Kingdom: Parthenon Press; 1999:378–88. Prapas Y, Prapas N, Hatziparasidou A, Prapa S, Nijs M, Vanderzwalmen P, et al. The echoguide embryo transfer maximizes the IVF results. Acta Eur Fertil 1995;26:113–5. Trokoudes KM, Pavlides C, Zhang X. Comparison outcome of fresh and vitrified donor oocytes in an egg-sharing donation program. Fertil Steril 2011; 95:1996–2000. Vanderzwalmen P, Zech N, Prapas Y, Panagiotidis Y, Papatheodorou A, Lejeune B, et al. Closed carrier device: a reality to vitrify oocytes and embryos in aseptic conditions [article in French]. Gynecologie Obstetrique Fertil 2010; 38:541–6. Bielanski A, Nadin-Davis S, Sapp T, Lutze-Wallace C. Viral contamination of embryos cryopreserved in liquid nitrogen. Cryobiology 2000;40:110–6. Bielanski A, Bergeron H, Lau PCK, Devenish J. Microbial contamination of embryos and semen during long term banking in liquid nitrogen. Cryobiology 2003;46:146–52. Official Journal of the European Union. Directive 2004/23/EC of the European Parliament and of the Council of 31 March 2004 on setting standards of quality and safety for the donation, procurement, testing, processing, preservation, storage and distribution of human tissues and cells EUR-LexL:2004:102:TOC-EN-EUR-Lex. Available at: http://eur-lex.europa.eu/legalcontent/EN/TXT/?uri¼OJ:L:2004:102:TOC. Accessed March 29, 2016. Official Journal of the European Union. Commission Directive 2006/17/EC of 8 February 2006 implementing Directive 2004/23/EC of the European Parliament and of the Council as regards certain technical requirements for the donation, procurement and testing of human tissues and cells. EUR-Lex-L:2006:038:TOC-EN-EUR-Lex. Available at: http://eur-lex.euro pa.eu/legal-content/EN/TXT/?uri¼OJ:L:2006:038:TOC. Accessed March 29, 2016. Lin L, Yu Y, Zhang X, Yang H, Bolund L, Callesen H, et al. Aseptic minimum volume vitrification technique for porcine parthenogenetically activated blastocyst. Cryo Letters 2011;32:62–8. Parmegiani L, Accorsi A, Cognigni GE, Bernardi S, Troilo E, Filicori M. Sterilization of liquid nitrogen with ultraviolet irradiation for safe vitrification of human oocytes or embryos. Fertil Steril 2010;94:1525–8. Eum JH, Park JK, Lee WS, Cha KR, Yoon TK, Lee DR. Long-term liquid nitrogen vapor storage of mouse embryos cryopreserved using vitrification or slow cooling. Fertil Steril 2009;91:1928–32. Yan J, Suzuki J, Yu X-M, Qiao J, Kan FWK, Chian R-C. Effects of duration of cryo-storage of mouse oocytes on cryo-survival, fertilization and embryonic development following vitrification. J Assist Reprod Genet 2011;28: 643–9.
VOL. - NO. - / - 2016
How does closed system vitrification of human oocytes affect the clinical outcome? A prospective, observational, cohort, noninferiority trial in an oocyte donation program Achilleas Papatheodorou, Ph.D.,a,b Pierre Vanderzwalmen, Ph.D.,c,d Yannis Panagiotidis, Ph.D.,a Stamatios Petousis, M.D., Ph.D.,a,e Giuseppe Gullo, M.D.,f Evangelia Kasapi,a Maria Goudakou, Ph.D.,a Nikos Prapas, M.D., Ph.D.,a Kostas Zikopoulos, M.D., Ph.D.,b Ioannis Georgiou, Ph.D.,b and Yannis Prapas, M.D., Ph.D.a a IAKENTRO Advanced Medical Center, Thessaloniki, Greece; b Center for Reproductive Medicine, Obstetrics and Gynecology, Ioannina University, Ioannina, Greece; c IVF Centers Prof Zech, Bregenz, Austria; d Chirec, Brussels, Belgium; e 3rd Department of Obstetrics and Gynaecology, Aristotle University, Thessaloniki, Greece; and f Department of Obstetrics and Gynaecology, University of Messina, Messina, Italy
Objective: To evaluate whether is possible to vitrify oocytes in an aseptic (hermetically closed) fashion and maintain clinical results comparable with those of fresh oocytes. Design: Prospective, observational, cohort, noninferiority trial. Setting: Private in vitro fertilization center. Patient(s): One hundred eighty-four recipients of donated vitrified oocytes. Intervention(s): Closed system vitrification. Main Outcome Measure(s): Pregnancy rate per cycle and clinical pregnancy rate per cycle. Result(s): No statistically significant differences were observed between two groups regarding the pregnancy rate per cycle (63.1% vs. 60.9%) or the clinical pregnancy rate per cycle (55.4% vs. 58.7%). Biochemical pregnancy rate was statistically significantly higher in the fresh group (7.6% vs. 2.2%). The mean number of embryos transferred was similar (2.0 0.0 vs. 1.97 0.3). Concerning embryologic data, there were no statistically significant differences regarding the fertilization, cleavage, top quality day-3 embryo, or blastocyst rates, whereas the top quality blastocyst rate on day 5 was statistically significantly higher in the fresh oocyte group (31.7% vs. 26.1%). Conclusion(s): Aseptically (in a closed system) vitrified oocytes show similar clinical efficiency compared with their sibling fresh oocytes. (Fertil SterilÒ 2016;-:-–-. Ó2016 by American Society for Reproductive Medicine.) Key Words: Aseptic technique, closed system vitrification, egg donor bank, oocyte donation, oocyte vitrification Discuss: You can discuss this article with its authors and with other ASRM members at https://www.fertstertdialog.com/users/16110fertility-and-sterility/posts/11072-how-does-closed-system-vitrification-of-human-oocytes-affect-the-clinical-outcome-a-prospectiveobservational-cohort-noninferiority-trial-in-an-oocyte-donation-program
F
or over 15 years vitrification as a cryopreservation technique has been successfully applied to
human embryos. From the moment that vitrification was proven to be a promising alternative to cryopreserve
Received February 5, 2016; revised June 17, 2016; accepted July 11, 2016. A.P. has nothing to disclose. P.V. has nothing to disclose. Y.P. has nothing to disclose. S.P. has nothing to disclose. G.G. has nothing to disclose. E.K. has nothing to disclose. M.G. has nothing to disclose. N.P. has nothing to disclose. K.Z. has nothing to disclose. I.G. has nothing to disclose. Y.P. has nothing to disclose. Reprint requests: Achilleas Papatheodorou, Ph.D., IAKENTRO Fertility Center, Agiou Vasileiou 4, 54250, Thessaloniki, Greece (E-mail: [email protected]). Fertility and Sterility® Vol. -, No. -, - 2016 0015-0282/$36.00 Copyright ©2016 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2016.07.1066 VOL. - NO. - / - 2016
oocytes, the popularity of the technique in the field of assisted reproduction technology (ART) has grown (1, 2). Many published studies have reported the superiority of vitrification over the slow freezing technique for oocyte cryopreservation, and the success rates using vitrified oocytes have been similar to those obtained with fresh oocytes (2–13). The birth of healthy infants resulting from vitrified oocytes (14–17) has established vitrification as 1
ORIGINAL ARTICLE: ASSISTED REPRODUCTION the gold standard technique for oocyte cryopreservation and has widened the indications for use of this technique. In the past, regardless the disappointing results, oocyte cryopreservation was the only option in specific circumstances, such as for women at risk of losing their ovarian reserve (from cancer, immunologic or genetic disorders, or aggressive medical treatments), for overcoming legal, ethical, and religious restrictions, or for addressing logistic situations such as an absent semen sample on the day of oocyte pickup. At present, due to the promising results and the safety of the technique, as demonstrated in healthy infants, vitrification is no longer considered an experimental method (18). With the introduction of efficient, harmless, and safe vitrification techniques, the indications for vitrifying oocytes could be widened and proposed to women who are seeking fertility preservation for social reasons or used to promote the development of egg donor banks. Noticeable is the fact that a longer period of storage, ranging from months to several years, will be needed if the demand for egg freezing increases in the future (19). In such prolonged storage conditions, safety is another key issue because the biological sample must be aseptically isolated and not in direct contact with liquid nitrogen (20). To guarantee aseptic vitrification and storage conditions, closed carrier systems have been introduced (21, 22). At present, few data using closed devices that ensure aseptic cooling and storage have been reported in cases of oocytes cryopreservation. Recent reviews (1, 23) have noted the obvious preference of scientists for using open systems for oocyte vitrification. This reluctance to vitrify using closed devices derives from a common belief (2, 23–25) that reduced cooling rates, such as those produced in closed systems due to thermo-isolation, could be harmful or lethal to the cells, increasing the probability of ice crystal formation during the cooling process (26). However, it has been reported that reduced cooling rates do not compromise survival rates in aseptically vitrified oocytes (27–30), zygotes (30, 31), or blastocysts (21, 32, 33) if very high warming rates are applied. Such statements are reinforced by recent studies of Mazur et al. (34–37), who have shown that the primary cause for cell injury or cell death during vitrification procedure is not the ice crystal formation during cooling but the recrystallization during warming (devitrification). According to their studies, warming rates are as important as cooling rates; in fact, warming rates should be higher than cooling rates for a successful vitrification and warming cycle. Therefore, a closed system could be as sufficient as an open one so long as we keep the warming rates high. So far, a few studies have described the competence of closed systems for oocyte vitrification (5, 27, 32, 38), but their clinical data are not enough to support the efficiency of these devices. Unlike the open systems, there are no prospective studies comparing fresh embryo transfers (ET) versus ET after oocyte vitrification in hermetically closed devices. To evaluate the efficiency of hermetically closed devices in oocyte vitrification we compared the clinical 2
outcome after ET derived from sibling oocytes to recipients being synchronized (fresh oocytes) or not (aseptically vitrified oocytes) with their donor in our oocyte donation program.
MATERIALS AND METHODS A prospective, observational, cohort study was performed at IAKENTRO Fertility Center from recruitment start date on January 19, 2014, to the completion date on December 15, 2014. All procedures were performed at the same laboratory. This clinical trial was a part of a doctoral study at the Medical School of University of Ioannina, Greece. The study was approved by the institutional review board of the Ioannina Medical School (Ref: 808a/8-3-2011). This trial was registered in ISRCTN registry (identification number ISRCTN56275481) and was approved by the IAKENTRO review board (reference number 1/2014, 19/1/2014). Informed consent was obtained from all women participating.
Noninferiority Test, Sample Size, and Study Design Based on a positive hCG/transfer baseline rate of 61% among controls and 59% for study subjects, a sample size of 92 transfers per arm would be required to be able to reject the null hypothesis that the one-sided 95% confidence interval (or equivalently a 90% two-sided confidence interval) will exclude a difference in favor of the standard group of more than 20% with a statistical significance level of 5% and a power (1-b) of 80%. Power calculation was performed with Sealed Envelope Ltd 2012 (39). Ninety-two oocyte donors participated in our study. A single stimulation cycle was included for each donor. Pairs of recipients, sharing sibling oocytes from the same donor, were included in the study. Each pair consisted of a recipient for whom fresh oocytes were used for their donation cycle and another recipient for whom the oocytes were vitrified and used after a short period of time. A single donation cycle was included for each recipient. One hundred and eightyfour couples who received sibling oocytes donated from the same donor were allocated to receive fresh (92 couples) or vitrified sibling oocytes (92 cases). Biological and clinical parameters were evaluated. Pregnancy rates were a secondary tracked outcome; the intervention did not depend on it, nor did it affect the execution of the study in any way. The current study contains a randomization procedure. During the donor's oocyte pickup, two separate dishes for oocyte collection were used. The retrieved cumulus oocyte complexes were randomly and equally assigned into the two dishes during oocytes retrieval. Odd numbered oocytes were allocated to the fresh group (dish 1: group 1), and even numbered oocytes were allocated to the closed vitrified group (dish 2: group 2). This allocation method could potentially have the drawback of recruiting a higher number of oocytes to group 1 as in all odd-numbered cases group 1 would have more oocytes enrolled. However, according to our experience, the larger follicles are first retrieved in each procedure are more likely to contain higher quality (mature) oocytes, so it is likely that the last-retrieved odd oocytes would be of poor VOL. - NO. - / - 2016
Fertility and Sterility® quality (immature) and would not finally result in a significantly different number of metaphase 2 (MII) oocytes in each group. Furthermore, it would demand an additional randomization process to decide which cases' oocyte allocation would begin from group 1 and from group 2. Therefore, to avoid complexity of procedure and because of the profound lack of clinical impact, it was initially decided to recruit oocytes in the same manner for all cases. Fresh group oocytes (group 1) were fertilized the same day, and vitrified group oocytes (group 2) were stored in liquid nitrogen tanks. The warming and the fertilization of group 2 oocytes were performed when the recipient to whom those oocytes had been allocated had her endometrium properly prepared.
Inclusion Criteria A detailed medical history of the donors was taken. The oocyte donors were %32 years old, had body mass index <30 kg/m2, regular menstrual cycles of 25–35 days, two normal ovaries based on transvaginal scan findings, no polycystic ovary syndrome, no known endometriosis, and no gynecologic or medical disorders. They all had agreed to donate their oocytes for treatment anonymously and altruistically. Oocyte donors were of known fertility and good ovarian response. Blood samples were collected for karyotyping and screening for previous viral infections (hepatitis B and C, human immunodeficiency virus, syphilis), thalassemia, and cystic fibrosis. Our study included patients with a minimal of eight mature oocytes, which is a basic requirement offered to recipient couples in our donation program. Consequently, after the denudation of retrieved oocytes, if a recipient wound up with %7 mature oocytes, these cycles were excluded from our analysis because one of the two recipient cycles had to be canceled. A total of 184 recipients, matched with their donors (n ¼ 92), were included in the study. All recipients were <50 years old with no history of endometriosis, and this was their first oocyte donation cycle. The recipients and their partners underwent blood screening similar to the donors, and a hysterosalpingogram and a diagnostic hysteroscopy eliminated cases presenting hydrosalpinx or intrauterine-related pathology. The recipients went through a mock transfer in a cycle before their donation cycle; if any difficulty was encountered, a cervical dilatation was performed (40). In both groups, the recipient couples had no form of severe male infertility indications. All the enrolled cases had normal values for concentration, motility, and morphology. The World Health Organization criteria (41) were used to evaluate the sperm quality. For each oocyte donation cycle, a recipient was allocated to receive the fresh and another to receive the vitrified oocytes, with an identical procedure: our center has a list of patients available for fresh oocytes and another list of patients available only for vitrified oocytes. Among all patients who had the same blood type and similar morphologic characteristics with the oocyte donor, there was one recipient from each list allocated by computerized random select software for each oocyte donation cycle. VOL. - NO. - / - 2016
Stimulation Protocol and Recipient Preparation The ovarian stimulation of the donors was performed with a fixed day-6 gonadotropin-releasing hormone (GnRH) antagonist protocol (42). Recipients in the fresh group underwent endometrial preparation as previously described elsewhere (43). Women with ovarian function were first downregulated in the luteal phase with a single-dose of GnRHagonist depot (Arvekap, 3.75 mg) beginning on day 21 of the previous cycle. One day after the announcement of the donor's period onset, the recipients were instructed to begin administering estradiol valerate (Cyclacur or Progynova) at 2 mg/d for the first 4 days, 4 mg/d for days 5–8, and 6 mg/d until the pregnancy test. The afternoon of the oocyte donation pickup, the recipients were administered 200 mg of progesterone (Utrogestan) intravaginally; this continued as 200 mg, three times a day, until a fetal heartbeat was observed by ultrasound. The recipients without menstruation followed the same protocol without the GnRH agonist. Endometrial development was evaluated by ultrasound scan; it was considered mature when the endometrial thickness was >9 mm. Recipients in the vitrification group followed the same treatment as fresh group recipients. They began with a GnRH agonist on the day 21 of the previous cycle. After confirming down-regulation by measuring serum estradiol and progesterone concentrations, we administered estrogen as mentioned previously. Endometrial development was evaluated by ultrasound scan; it was considered mature when the endometrial thickness was >9 mm. If the endometrium was <9 mm, the transfer cycle was canceled. The embryos of the fresh oocyte group were vitrified on day 5, and the transfer was postponed for a future cycle. These cycles and their sibling cases were not included in our cohort. In vitrified oocyte group, when the endometrium was <9 mm, the oocytes were not warmed, and the cycle was postponed for a future date (with new endometrial preparation).
Laboratory Procedures The oocytes retrieved from each donor were equilibrated in Single Step Medium (SSM; Irvine Scientific) for 2 hours and then enzymatically denuded (hyaluronidase solution; Irvine Scientific). Only MII oocytes were included in the study groups. Fresh group (group 1) oocytes were injected 1 hour after denudation. Closed vitrification group (group 2) oocytes were vitrified a half-hour to 1 hour after denudation. After their warming, oocytes were cultured in SSM for 3 hours before they were injected. Intracytoplasmic sperm injection (ICSI) was performed in all cases. Fertilization was assessed 16 to 20 hours after ICSI by visualization of the two pronuclei. Embryos were cultured in 30-mL droplets of SSM, overlaid with mineral oil (FertiCult Mineral Oil - FERTIPRO) in a MINC benchtop incubator (Cook Medical) with provided triple gas of 6% CO2, 5% O2, and 89% N2. Embryo quality was assessed on days 3 and 5. For day-3 embryos, the number of cells, the appearance of blastomeres, and the presence of cytoplasm defects or fragmentation were evaluated (44). According to this system, top quality cleaved embryos were considered those with 8 to 12 symmetric blastomeres with absence of cytoplasm defects and without or with negligible fragmentation. 3
ORIGINAL ARTICLE: ASSISTED REPRODUCTION For the evaluation of day-5 embryos we used the Gardner and Schoolcraft criteria (45) in which thin zona pellucida, smooth trophectoderm, equality and close adhesion of blastomeres, clearly visible blastocyst cavity, and well-developed inner cell mass with many closely aggregated cells are the most important parameters correlating to top blastocyst quality. We performed ET performed on day 5 under ultrasound guidance as previously described elsewhere (46). The remaining embryos were vitrified on day 5 using a closed vitrification system.
Oocyte Vitrification in a Closed System Oocytes were vitrified by using the Vitrisafe carrier (21) (VitriMed) at a maximum of 3 hours after oocyte pickup. Denuded oocytes were exposed to four equilibration solutions with 1.25%, 2.5%, 5%, and 10% concentrations of dimethyl sulfoxide and ethylene glycol (FertiVit Cooling Kit - FERTIPRO) for 3, 3, 3, and 5:30 minutes, respectively, and one vitrification solution with 20% dimethyl sulfoxide and 20% ethylene glycol plus 0.75 mol/L sucrose and 10 mg/L Ficoll (FertiVit Warming Kit - FERTIPRO) for 1 minute. After exposure to the cryoprotectants, by means of a single drop the oocytes were initially transferred on the Vitrisafe, and then the carrier was inserted in the protective straw, which was thermosealed and plunged into a Dewar with liquid nitrogen. All procedures were performed at room temperature (25 C). Warming was performed in five steps as described elsewhere (28). Briefly, the oocytes were exposed to gradually decreased concentrations—1 M, 0.75 M, 0.5 M, 0.25 M, and 0.125 M—of sucrose for 1, 1, 2, 2, and 2 minutes, respectively (Fertipro). The rehydration of the oocytes was performed at room temperature (25 C) with an exception of the first step, in which the Vitrisafe was immersed in a warm solution (37 C) of 1 M sucrose for 1 minutes. To recover, the oocytes were placed into culture medium (SSM) and incubated for 3 hours at 37 C (6% CO2 and 5% O2) before they were injected. Degenerated oocytes were removed from the cohort.
Outcome Measures The number of surviving oocytes, fertilized oocytes, cleavage embryos, top quality cleavage embryos, blastocysts, top quality blastocysts, and embryos transferred were analyzed. Oocyte survival rate was defined as the number of oocytes that survived out of the total number of oocytes warmed. We applied ICSI with all surviving oocytes. Fertilization rate was defined as the number of fertilized oocytes out of the number of oocytes that survived. Cleavage rate, top cleavage rate, blastocyst rate, and top quality blastocyst rate were defined as the number of cleavage embryos, top quality cleavage embryos, blastocysts, and top quality blastocysts, respectively, out of the total number of mature (MII) oocytes. Pregnancy was confirmed by the rise of serum b-human chorionic gonadotropin (hCG) concentration 14 days after ET. Clinical pregnancy was defined by the appearance of a gestational sac and a fetal heartbeat at 8 to 10 weeks of gestation. 4
Ongoing pregnancy rate was considered the number of pregnancies with fetuses displaying heart activity beyond 12 weeks of gestation per cycle. The miscarriage rate was considered the loss of a clinical pregnancy per cycle before 20 completed weeks of gestation. Implantation rate was defined as the number of gestational sacs per transferred embryos. As biochemical pregnancy was considered the initial rise and the immediate fall of hCG in serum level verified by the absence of a gestational sac under the ultrasound. As a private in vitro fertilization (IVF) center and not a reference center or a public hospital, we did not monitor the couples in the study by ourselves. Live births were reported to our clinic after communication with the couple or their gynecologist. Babies who had no reports of anomalies were considered as healthy.
Primary and Secondary Outcomes The primary end point of the study was the positive hCG rate per cycle. Secondary outcomes were clinical pregnancy rate per cycle, ongoing pregnancy rate, biochemical pregnancy rate, miscarriage rate, twinning rate, and live-birth rate. Secondary end points were also the oocyte survival rate, the fertilization rate, the cleavage and top cleavage rates, and the blastocyst and top blastocyst rates.
Statistical Analysis Continuous data are expressed as mean standard deviation, and categorical data as percentages (%). Normality was assessed with the Kolmogorov-Smirnov test. An independent sample t test was used for parametric and Mann-Whitney test for nonparametric numeric variables. Categorical data were analyzed with chi-square analysis. The statistical analyses were performed using the Statistical Package for Social Sciences, version 17.0 (IBM/SPSS Inc.). The non-inferiority test was performed by using the power calculator of sealed envelope at www.sealedenvelope.com.
RESULTS Data from 184 recipients were prospectively collected from January 2014 to December 2014. No cases were included in the study who finally dropped out due to survival, fertilization, or blastocyst formation failures. The epidemiologic characteristics of the included cases were similar between the two groups (Table 1). A total of 2,325 oocytes were retrieved: 1,175 in group 1, and 1,150 in group 2. Of these, 1,966 of were MII: 982 in the fresh group and 984 in the closed vitrification group (Table 2). There was no difference in the number of the allocated oocytes between the two groups (10.7 2.1 vs. 10.7 2.0, P¼ .94), thus confirming the initial hypothesis that our oocyte allocation method would not result in a statistically significantly different number of MII oocytes. The survival rate of the oocytes from group 2 was 92.7% (912 of 984 oocytes). No statistically significant difference was observed regarding the fertilization rate, cleavage rate, top quality embryos on day 3, or blastocyst rate between the two groups. The top quality blastocyst rate on day 5 was statistically significantly VOL. - NO. - / - 2016
Fertility and Sterility®
TABLE 1 Baseline characteristics of donors, recipients, and the recipients' male partners. Characteristics Donor Age (y) BMI (kg/m2) Stimulation (d, range) Total rFSH (IU) E2 (pg/mL) Recipient Age (y) Endometrium thickness BMI (kg/m2) Endometrial preparation (d) Male partner Age (y) Sperm concentration (millions/mL) Motility Forward motility Morphology
Donor
Group 1 fresh oocytes (n [ 92)
Group 2 vitrified oocytes (n [ 92)
P value
43.2 3.4 10.9 1.1 23.8 3.5 16.9 1.1
43.5 3.4 10.7 0.9 23.6 3.1 16.8 0.5
.57 .31 .61 .42
43.62 5.6 49.2 22.6 67.87 11.8 56.1 13.1 16.52 4.6
42.92 5.7 46.6 22.4 67.5 11.8 56.6 14.3 16.43 6.4
.41 .42 .85 .84 .91
28 3.2 22.6 2.6 9.8 1.1 (8–12) 2,192 407 2,230 640
Note: Values are mean standard deviation. BMI ¼ body mass index; E2 ¼ estradiol; rFSH ¼ recombinant follicle-stimulating hormone. Papatheodorou. Comparison of fresh and vitrified oocytes. Fertil Steril 2016.
higher in the fresh oocyte group (31.7% vs. 26.1%, P¼ .003). Embryologic parameters are presented in Table 2. Ninety-two transfers were performed in each group. The mean number of embryos transferred was similar (2.0 0.0 vs. 1.97 0.3, P¼ .25). No statistically significant difference was observed between two groups regarding pregnancy (bhCG positive) rate per cycle (63.1% vs. 60.9%, P¼ .76) or clinical pregnancy rate per cycle (55.4% vs. 58.7%, P¼ .66). No statistically significant differences were observed regarding all the other outcomes between the two groups. Regarding live births, 66 healthy babies were born from the fresh group and 62 from the vitrification group. No adverse outcome was reported by the parents. The primary
and secondary clinical outcomes of the present study are presented in Table 3. We have calculated that the collected oocytes to baby ratio was 5.6% and the MII oocytes to baby ratio was 6.8% in the fresh group. In the vitrification group the ratios were 5.3% and 6.3%, respectively (Table 4). The MII oocytes needed to achieve a pregnancy were 14.8 in fresh group and 15.8 in the vitrification group (Table 4). The power analysis according to observed rates (63.1% for controls and 60.9% for patients) indicated that, with 92 women in each group, our study had a 80.5% power to reject the null hypothesis that one-sided 95% confidence interval (or equivalently a 90% two-sided confidence interval) will
TABLE 2 Comparison of the biological parameters of group 1 (fresh) and group 2 (vitrified). Parameters Retrieved oocytes MII oocytes Vitrified oocytes Survived oocytes Survival rate, % Fertilized oocytes Fertilization rate, % Cleaved embryos (d 3) Cleavage rate, % Top quality cleaved embryos Top quality cleavage rate, % Blastocysts Blastocyst rate, % Top quality blastocyst Top quality blastocyst rate, % Embryos transferred Vitrified embryos
Group 1 fresh oocytes (n [ 92)
Group 2 vitrified oocytes (n [ 92)
P value
12.7 2.4 (1,175) 10.7 2.1 (982) – – – 8.6 1.8 (796) 81.1% (796/982) 8.0 1.6 (739) 75.2% (739/982) 4.3 2.0 (403) 41.1% (403/982) 5.2 1.7 (484) 49.3% (484/982) 3.4 2.0 (312) 31.7% (312/982) 2.0 0.0 (184) 2.76 1.5 (254)
12.5 2.1 (1,150) 10.7 2.0 (984) 10.7 2.0 (984) 9.9 2.1 (912) 92.7% (912/984) 8.1 1.7 (744) 81.6% (744/912) 7.5 2.0 (692) 75.8% (692/912) 4.1 2.1 (382) 41.8% (382/912) 4.8 2.1 (440) 48.2% (440/912) 2.6 1.6 (238) 26.1% (238/912) 1.96 0.4 (180) 2.41 1.8 (222)
.86 .94 – – – .23 .77 .29 .90 .37 .80 .10 .51 .003 .003 .25 .16
Note: Values are mean standard deviation (number) unless otherwise indicated. Papatheodorou. Comparison of fresh and vitrified oocytes. Fertil Steril 2016.
VOL. - NO. - / - 2016
5
ORIGINAL ARTICLE: ASSISTED REPRODUCTION
TABLE 3 Number of embryos transferred and clinical parameters per cycle.
Parameters Positive b-hCG Clinical pregnancy rate Ongoing pregnancy rate Implantation rate Biochemical pregnancy rate Miscarriage rate Delivery rate Twin pregnancy rate Live births
Group 1 fresh oocytes (n [ 92)
Group 2 vitrified oocytes (n [ 92)
P value
63.1% (58/92) 55.4% (51/92)
60.9% (56/92) 58.7% (54/92)
.76 .66
51.1% (47/92)
52.2% (48/92)
.88
41.8% (77/184) 7.6% (7/92)
38.9% (70/180) 2.2% (2/92)
.57 .05
4.3% (4/92) 51.1% (47/92) 40.4% (19/47) 66
6.5% (6/92) 52.1% (48/92) 29.2% (14/48) 62
.52 .88 .25 .23
Note: b-hCG ¼ b-human chorionic gonadotropin. Papatheodorou. Comparison of fresh and vitrified oocytes. Fertil Steril 2016.
exclude a difference in favor of the standard group of more than 20%.
DISCUSSION Our prospective comparative study shows the parity of fresh and sibling vitrified oocytes. No differences were observed regarding the pregnancy rate, clinical pregnancy rate, ongoing pregnancy rate, or live-birth rate between the fresh and vitrified oocytes. Additionally, it has been shown that fresh and sibling vitrified oocytes have similar developmental potential and implantation capacity. The only statistically significant differences we obtained were a higher top quality blastocyst rate on day 5 in favor of the fresh group and a lower biochemical pregnancy rate in favor of the vitrification group. In this study, to eliminate confounding effects related to female age and ovarian stimulation, a population of oocyte donors and their sibling recipients who shared the oocytes of the same donor were studied. Furthermore the design of the current study shows several strengths. To our knowledge, this work is the only prospective study with the following characteristics: [1] prospective study with randomization, [2] sibling donated oocytes, [3] one fresh group and one vitri-
TABLE 4 Oocyte to baby ratio and oocytes used to achieve a pregnancy.
Parameter Collected oocytes to baby ratio Collected oocytes used to achieve a live birth rate MII oocytes to baby ratio MII oocytes used to achieve a live birth rate
Group 1 fresh oocytes (n [ 92)
Group 2 vitrified oocytes (n [ 92)
5.6% 17.8
5.3% 18.5
6.8% 14.8
6.3% 15.8
Note: MII ¼ metaphase 2. Papatheodorou. Comparison of fresh and vitrified oocytes. Fertil Steril 2016.
6
fication group, [4] vitrification of oocytes performed in a closed system (aseptic conditions), and [5] blastocyst culture and transfer performed in both groups. Because of these characteristics, we consider our findings of high importance. The ultimate proof of vitrified oocyte competency is to achieve comparable live-birth rates to fresh oocytes. Using the open vitrification technique in an oocyte donation program Cobo et al., obtained similar outcomes between fresh and vitrified oocytes (11). Later, Trokoudes et al. (47) reported comparable results obtained with vitrified oocytes using the open technique. Our study in an oocyte donation program has shown that the same results can be achieved with a closed vitrification device because the implantation, pregnancy, clinical pregnancy, and ongoing pregnancy rates were no different between the vitrified and fresh sibling oocytes. Cobo et al. (19) have reported on the oocyte to baby ratio (6.5%: 6.5 babies born for every 100 oocytes used) and the number of oocytes needed (15.4) to achieve a baby. These results are taken from a large database of 32,460 heterologous and 1,513 autologous oocytes. In our study the collected oocytes to baby ratio was 5.6% and the MII oocytes to baby ratio was 6.8% in the fresh group; in the vitrification group the ratios were 5.3% and 6.3%, respectively (Table 4). The MII oocytes needed to achieve a pregnancy were 14.8 in fresh group and 15.8 in the vitrification group (Table 4). In both studies, we see that a relatively big number of MII oocytes (around 15) are needed to achieve a live birth. In fact, Cobo et al. (19) report that the more oocytes used, the higher the cumulative pregnancy rate. When five oocytes are used the rate is low, and it statistically significantly increases as the number of consumed oocytes doubles. It is very important to notice that these numbers are referring to oocytes donated from young healthy donors. We must be very careful to not extrapolate the above ratios to women of different age groups or different fertility statuses. The oocyte to baby ratio would be completely different for these women, so we must be careful when we advise them to cryopreserve their oocytes. This is something that is mentioned by Cobo et al. (19), who calculated that women over 40 years old should consume 55.5 oocytes to achieve the live-birth rate. Looking at the vitrification group we observed a survival rate of 92.7% after warming. This result is similar to those reported in open vitrification system studies (11), and the average reported in a recent systematic review and metaanalysis (1). The fertilization, cleavage, top cleavage, and blastocyst rates were similar between the vitrified oocytes and fresh groups and in accordance with recent studies that have used vitrified donor oocytes (1). The scope of our study was not to compare closed and open vitrification devices, but comparing the efficiency of the devices was inevitable. The parity of closed versus open vitrification devices has been well known to our team since 2009 (21, 48) and is well established by randomized, controlled trials in embryos (32) and oocytes (33). Nonetheless, scientists have an obvious preference for open system devices (1, 23) due to a common belief that high cooling rates, accomplished by direct contact of the sample with liquid nitrogen, are the key point to successful oocyte vitrification. This belief was challenged by Seki and Mazur VOL. - NO. - / - 2016
Fertility and Sterility® (34–36) who proved that warming rates are of equal or higher importance as the cooling rates. They showed that once a certain high warming rate is achieved, the initial cooling rate is of less importance; if you warm fast enough, the cells will survive. Our study has confirmed their findings because the closed devices used for the vitrification could maintain the clinical efficiency of the vitrified oocytes. It was shown that although the cooling rates of closed system devices are lower than the equivalent of open system, they are still high enough and in combination with a very rapid warming procedure were able to obtain excellent survival rates and demonstrate the competency of the vitrified oocytes. The development of a reliable and safe aseptic (closed) vitrification protocol is of high importance for human tissue cryopreservation, especially after the Bielanski's reports on possibility of cross-contamination under liquid nitrogen that have raised skepticism on the use of open vitrification protocols (49, 50). The guidelines of European Parliament (European Parliament and the Council of the European Union, 2004, 2006) has impelled scientist to look for solutions that would maintain vitrification in an aseptic status (51, 52)—for instance, liquid nitrogen sterilized by filtration (53) or UV irradiation (54) or the storage of samples in vapors of nitrogen (25, 55). The greatest question has been how to solve the problem using the open system devices. The closed systems were examined and considered as potentially harmful for the cells due to the lower cooling rates. The results of our study have shown that closed vitrification systems are sufficient and can be used as devices to store biological samples in the safest way. At present, with an increase of social egg freezing and oocyte donation banking, we can expect a longer storage period for these oocytes, ranging from some months to several years. In such prolonged conditions our main concern is to store biological material in the safest way. A closed system device could guarantee the appropriate isolation from any detrimental factors such as the toxic low-molecular-weight compounds found in liquid nitrogen (56) whose long-term actions are not known. Our prospective comparative study sends an important message: vitrified oocytes have similar clinical efficiency to fresh oocytes. Additionally, their efficiency can be maintained even if we use a closed system for vitrification, which leaves behind any concerns regarding the biosafety of vitrified human tissue.
REFERENCES 1.
2.
3.
4.
Potdar N, Gelbaya TA, Nardo LG. Oocyte vitrification in the 21st century and post-warming fertility outcomes: a systematic review and meta-analysis. Reprod Biomed Online 2014;29:159–76. Kuwayama M, Vajta G, Kato O, Leibo SP. Highly efficient vitrification method for cryopreservation of human oocytes. Reprod Biomed Online 2005;11:300–8. Cao Y-X, Xing Q, Li L, Cong L, Zhang Z-G, Wei Z-L, et al. Comparison of survival and embryonic development in human oocytes cryopreserved by slowfreezing and vitrification. Fertil Steril 2009;92:1306–11. Rezazadeh Valojerdi M, Eftekhari-Yazdi P, Karimian L, Hassani F, Movaghar B. Vitrification versus slow freezing gives excellent survival, post
VOL. - NO. - / - 2016
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23. 24.
warming embryo morphology and pregnancy outcomes for human cleaved embryos. J Assist Reprod Genet 2009;26:347–54. Smith GD, Serafini PC, Fioravanti J, Yadid I, Coslovsky M, Hassun P, et al. Prospective randomized comparison of human oocyte cryopreservation with slow-rate freezing or vitrification. Fertil Steril 2010;94:2088–95. Ubaldi F, Anniballo R, Romano S, Baroni E, Albricci L, Colamaria S, et al. Cumulative ongoing pregnancy rate achieved with oocyte vitrification and cleavage stage transfer without embryo selection in a standard infertility program. Hum Reprod 2010;25:1199–205. Cobo A, Diaz C. Clinical application of oocyte vitrification: a systematic review and meta-analysis of randomized controlled trials. Fertil Steril 2011; 96:277–85. J, Boada M, Clua E, Rodríguez I, Martínez F, et al. How does Sole M, Santalo vitrification affect oocyte viability in oocyte donation cycles? A prospective study to compare outcomes achieved with fresh versus vitrified sibling oocytes. Hum Reprod 2013;28:2087–92. Levi Setti PE, Porcu E, Patrizio P, Vigiliano V, de Luca R, d'Aloja P, et al. Human oocyte cryopreservation with slow freezing versus vitrification. Results from the National Italian Registry data, 2007–2011. Fertil Steril 2014;102: 90–5.e2. Cobo A, Kuwayama M, Perez S, Ruiz A, Pellicer A, Remohí J. Comparison of concomitant outcome achieved with fresh and cryopreserved donor oocytes vitrified by the Cryotop method. Fertil Steril 2008;89:1657–64. Cobo A, Meseguer M, Remohí J, Pellicer A. Use of cryo-banked oocytes in an ovum donation programme: a prospective, randomized, controlled, clinical trial. Hum Reprod 2010;25:2239–46. Cobo A, Garrido N, Crespo J, Jose R, Pellicer A. Accumulation of oocytes: a new strategy for managing low-responder patients. Reprod Biomed Online 2012;24:424–32. Rienzi L, Cobo A, Paffoni A, Scarduelli C, Capalbo A, Vajta G, et al. Consistent and predictable delivery rates after oocyte vitrification: an observational longitudinal cohort multicentric study. Hum Reprod 2012;27: 1606–12. Chian R-C, Huang JYJ, Tan SL, Lucena E, Saa A, Rojas A, et al. Obstetric and perinatal outcome in 200 infants conceived from vitrified oocytes. Reprod Biomed Online 2008;16:608–10. Cobo A, Serra V, Garrido N, Olmo I, Pellicer A, Remohí J. Obstetric and perinatal outcome of babies born from vitrified oocytes. Fertil Steril 2014;102: 1006–15.e4. Noyes N, Porcu E, Borini A. Over 900 oocyte cryopreservation babies born with no apparent increase in congenital anomalies. Reprod Biomed Online 2009;18:769–76. Cai L-B, Qian X-Q, Wang W, Mao Y-D, Yan Z-J, Liu C-Z, et al. Oocyte vitrification technology has made egg-sharing donation easier in China. Reprod Biomed Online 2012;24:186–90. Practice Committees of American Society for Reproductive Medicine, Society for Assisted Reproductive Technology. Mature oocyte cryopreservation: a guideline. Fertil Steril 2013;99:37–43. Cobo A, Garrido N, Pellicer A, Remohí J. Six years' experience in ovum donation using vitrified oocytes: report of cumulative outcomes, impact of storage time, and development of a predictive model for oocyte survival rate. Fertil Steril 2015;104:1426–34.e8. Wirleitner B, Vanderzwalmen P, Bach M, Baramsai B, Neyer A, Schwerda D, et al. The time aspect in storing vitrified blastocysts: its impact on survival rate, implantation potential and babies born. Hum Reprod 2013;28:2950–7. Vanderzwalmen P, Ectors F, Grobet L, Prapas Y, Panagiotidis Y, Vanderzwalmen S, et al. Aseptic vitrification of blastocysts from infertile patients, egg donors and after IVM. Reprod Biomed Online 2009;19:700–7. Van Landuyt L, Stoop D, Verheyen G, Verpoest W, Camus M, Van de Velde H, et al. Outcome of closed blastocyst vitrification in relation to blastocyst quality: evaluation of 759 warming cycles in a single-embryo transfer policy. Hum Reprod 2011;26:527–34. Vajta G, Rienzi L, Ubaldi FM. Open versus closed systems for vitrification of human oocytes and embryos. Reprod Biomed Online 2015;30:325–33. Nagy ZP, Chang C-C, Shapiro DB, Bernal DP, Kort HI, Vajta G. The efficacy and safety of human oocyte vitrification. Semin Reprod Med 2009;27: 450–5.
7
ORIGINAL ARTICLE: ASSISTED REPRODUCTION 25.
26. 27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
8
Cobo A, Romero JL, P erez S, de los Santos MJ, Meseguer M, Remohí J. Storage of human oocytes in the vapor phase of nitrogen. Fertil Steril 2010;94: 1903–7. Rall WF, Fahy GM. Ice-free cryopreservation of mouse embryos at 196 degrees C by vitrification. Nature 1985;313:573–5. Stoop D, De Munck N, Jansen E, Platteau P, Van den Abbeel E, Verheyen G, et al. Clinical validation of a closed vitrification system in an oocyte-donation programme. Reprod Biomed Online 2012;24:180–5. Papatheodorou A, Vanderzwalmen P, Panagiotidis Y, Prapas N, Zikopoulos K, Georgiou I, et al. Open versus closed oocyte vitrification system: a prospective randomized sibling-oocyte study. Reprod Biomed Online 2013;26:595–602. De Munck N, Verheyen G, Van Landuyt L, Stoop D, Van de Velde H. Survival and post-warming in vitro competence of human oocytes after high security closed system vitrification. J Assist Reprod Genet 2013;30:361–9. Schiewe MC, Zozula S, Anderson RE, Fahy GM. Validation of microSecure vitrification (mS-VTF) for the effective cryopreservation of human embryos and oocytes. Cryobiology 2015;71:264–72. Vanderzwalmen P, Zech NH, Ectors F, Stecher A, Lejeune B, Vanderzwalmen S, et al. Blastocyst transfer after aseptic vitrification of zygotes: an approach to overcome an impaired uterine environment. Reprod Biomed Online 2012;25:591–9. Panagiotidis Y, Vanderzwalmen P, Prapas Y, Kasapi E, Goudakou M, Papatheodorou A, et al. Open versus closed vitrification of blastocysts from an oocyte-donation programme: a prospective randomized study. Reprod Biomed Online 2013;26:470–6. Papatheodorou A, Panagiotidis Y, Kasapi E, Goudakou M, Passadaki T, Prapas N, et al. Fresh vs aseptic vitrified sibling embryo transfers in oocyte donation program: a prospective, observational, comparative study. Hum Reprod 2013;28(Suppl 1):i62–4. Seki S, Mazur P. The dominance of warming rate over cooling rate in the survival of mouse oocytes subjected to a vitrification procedure. Cryobiology 2009;59:75–82. Mazur P, Seki S. Survival of mouse oocytes after being cooled in a vitrification solution to 196 C at 95 to 70,000 C/min and warmed at 610 to 118,000 C/min: a new paradigm for cryopreservation by vitrification. Cryobiology 2011;62:1–7. Seki S, Mazur P. Ultra-rapid warming yields high survival of mouse oocytes cooled to 196 c in dilutions of a standard vitrification solution. PLoS One 2012;7:e36058. Jin B, Mazur P. High survival of mouse oocytes/embryos after vitrification without permeating cryoprotectants followed by ultra-rapid warming with an IR laser pulse. Sci Rep 2015;5:9271. Paffoni A, Guarneri C, Ferrari S, Restelli L, Nicolosi AE, Scarduelli C, et al. Effects of two vitrification protocols on the developmental potential of human mature oocytes. Reprod Biomed Online 2011;22:292–8. Sealed Envelope. Power calculator for binary outcome non-inferiority trial. Available at: https://www.sealedenvelope.com/power/binary-noninferior/. Accessed April 22, 2016. Prapas N, Prapas Y, Panagiotidis Y, Prapa S, Vanderzwalmen P, Makedos G. Cervical dilatation has a positive impact on the outcome of IVF in randomly assigned cases having two previous difficult embryo transfers. Hum Reprod 2004;19:1791–5. World Health Organization. Laboratory manual for the examination and processing of human semen. 5th ed. Geneva: WHO; 2010.
42.
43.
44.
45.
46.
47.
48.
49. 50.
51.
52.
53.
54.
55.
56.
Prapas N, Prapas Y, Panagiotidis Y, Prapa S, Vanderzwalmen P, Schoysman R, et al. GnRH agonist versus GnRH antagonist in oocyte donation cycles: a prospective randomized study. Hum Reprod 2005; 20:1516–20. Prapas N, Tavaniotou A, Panagiotidis Y, Prapa S, Kasapi E, Goudakou M, et al. GnRH antagonists and endometrial receptivity in oocyte recipients: a prospective randomized trial. Reprod Biomed Online 2009;18:276–81. Veeck LL. An atlas of human gametes and conceptuses: an illustrated reference for assisted reproductive technology. New York: Taylor & Francis; 1999. Gardner D, Schoolcraft W. In vitro culture of human blastocyst. In: Towards reproductive certainty: infertility and genetics beyond. Carnforth, United Kingdom: Parthenon Press; 1999:378–88. Prapas Y, Prapas N, Hatziparasidou A, Prapa S, Nijs M, Vanderzwalmen P, et al. The echoguide embryo transfer maximizes the IVF results. Acta Eur Fertil 1995;26:113–5. Trokoudes KM, Pavlides C, Zhang X. Comparison outcome of fresh and vitrified donor oocytes in an egg-sharing donation program. Fertil Steril 2011; 95:1996–2000. Vanderzwalmen P, Zech N, Prapas Y, Panagiotidis Y, Papatheodorou A, Lejeune B, et al. Closed carrier device: a reality to vitrify oocytes and embryos in aseptic conditions [article in French]. Gynecologie Obstetrique Fertil 2010; 38:541–6. Bielanski A, Nadin-Davis S, Sapp T, Lutze-Wallace C. Viral contamination of embryos cryopreserved in liquid nitrogen. Cryobiology 2000;40:110–6. Bielanski A, Bergeron H, Lau PCK, Devenish J. Microbial contamination of embryos and semen during long term banking in liquid nitrogen. Cryobiology 2003;46:146–52. Official Journal of the European Union. Directive 2004/23/EC of the European Parliament and of the Council of 31 March 2004 on setting standards of quality and safety for the donation, procurement, testing, processing, preservation, storage and distribution of human tissues and cells EUR-LexL:2004:102:TOC-EN-EUR-Lex. Available at: http://eur-lex.europa.eu/legalcontent/EN/TXT/?uri¼OJ:L:2004:102:TOC. Accessed March 29, 2016. Official Journal of the European Union. Commission Directive 2006/17/EC of 8 February 2006 implementing Directive 2004/23/EC of the European Parliament and of the Council as regards certain technical requirements for the donation, procurement and testing of human tissues and cells. EUR-Lex-L:2006:038:TOC-EN-EUR-Lex. Available at: http://eur-lex.euro pa.eu/legal-content/EN/TXT/?uri¼OJ:L:2006:038:TOC. Accessed March 29, 2016. Lin L, Yu Y, Zhang X, Yang H, Bolund L, Callesen H, et al. Aseptic minimum volume vitrification technique for porcine parthenogenetically activated blastocyst. Cryo Letters 2011;32:62–8. Parmegiani L, Accorsi A, Cognigni GE, Bernardi S, Troilo E, Filicori M. Sterilization of liquid nitrogen with ultraviolet irradiation for safe vitrification of human oocytes or embryos. Fertil Steril 2010;94:1525–8. Eum JH, Park JK, Lee WS, Cha KR, Yoon TK, Lee DR. Long-term liquid nitrogen vapor storage of mouse embryos cryopreserved using vitrification or slow cooling. Fertil Steril 2009;91:1928–32. Yan J, Suzuki J, Yu X-M, Qiao J, Kan FWK, Chian R-C. Effects of duration of cryo-storage of mouse oocytes on cryo-survival, fertilization and embryonic development following vitrification. J Assist Reprod Genet 2011;28: 643–9.
VOL. - NO. - / - 2016