Compared to Vitrification, Slow Freezing Technique Is Associated with a Higher Post-Thawed Embryos Survival and Clinical Pregnancy Rates

Central Medical Journal of Obstetrics and Gynecology

Research Article *Corresponding authors Mamdoh A Eskandar, Department of Obstetrics and Genecology and Reproductive Medicine King Khalid University, College of Medicine Abha, PO Box 641, Saudi Compared to Vitrification, Arabia, Tel: 996 7 2284635; Fax: 996 7 2257201;E-mail: Slow Freezing Technique is Submitted: 19 November 2014 Accepted: 03 December 2014 Published: 05 December 2014 Associated With a Higher Post- ISSN: 2333-6439 Copyright Thawed Embryos Survival and © 2014 Eskandar et al. Clinical Pregnancy Rates. Is This OPEN ACCESS Keywords • Slow Freezing a Myth or a Fact? • Vitrification • Cryopreservation Fawaz Edris1,2, Salma Baghdadi2, Ahmed Fares3, Warda • Embryo Survival Rate 6 4 4 5 • Human Chorionic Gonadotropin Alasmari , Suleiman Dabit , Susan Sousou , Aza Al-Yamani , • In vitro Fertilization Mamdoh Eskandar3,5 1Department of Obstetrics and Gynecology, Umm Al-Qura University, Saudi Arabia 2Department of Obstetrics and Gynecology, International Medical Center, Saudi Arabia 3Department of Obstetrics and Gynecology, Saudi Center for Assisted Reproduction, Saudi Arabia 4Department of Obstetrics and Gynecology Khalidi Medical center, Jordan 5Department of Obstetrics and Gynecology, King Khalid University, Saudi Arabia 6Department of Anatomy, Umm Al-Qura University, Saudi Arabia

Abstract Objective: To compare the survival and clinical pregnancy rates of post- thawed human embryos, following cryopreservation using slow freezing and vitrification techniques. Design: Retrospective cohort study. Setting: Private In-Vitro Fertilization (IVF) centre, Saudi Arabia. Patients: 516 patients who underwent IVF between June 2003 and June 2011 and had surplus embryos that were suitable for cryopreservation. Method: Outcomes of interest were compared between embryos cryopreserved at cleavage stage (luteal day-3) by either slow freezing (322 patients) or vitrification (194 patients) techniques. Results: The slow freezing group had significantly higher post-thaw embryo survival (69.75 vs. 62.75, P=0.012) and clinical pregnancy (34.8 vs. 21.5, P=0.002) rates. Conclusions: In our experience, slow freezing of human embryos at cleavage stage (luteal day-3) yielded better results than vitrification. Larger controlled trials are needed to support this conclusion.

INTRODUCTION postponement of embryo transfer (ET) to a future cycle feasible, thus decreasing the incidence of ovarian hyperstimulation Cryopreservation allows the transfer of limited number of syndrome (OHSS) in high-risk patients, while maintaining the fresh embryos back to the uterus while saving the remaining probability of pregnancy [1]. ones for future use; thus maximizing the cumulative effectiveness of an IVF cycle. Additionally, cryopreservation makes the The main problem during embryo cryopreservation

Cite this article: Edris F, Baghdadi S, Fares A, Alasmari W, Dabit S, et al. (2014) Compared to Vitrification, Slow Freezing Technique is Associated With a Higher Post-Thawed Embryos Survival and Clinical Pregnancy Rates. Is This a Myth or a Fact? Med J Obstet Gynecol 2(4): 1047. Eskandar et al. (2014) Email: Central is the formation of intracellular ice, which can lead to cell according to a standard scoring system and labelled as Grade I, damage and developmental arrest. To overcome this problem, different cryopreservation protocols, such as slow freezing and embryo quality/grade, a maximum of 3 embryos were selected andII, III culturedor IV. Depending for 20 on to the 120 patient minutes ̓s age, (min) previous in Embryo history, Glue and solutions, such as propanediol and dimethyl sulfoxide (DMSO), (Vitrolife , Sweden) prior to ET, which was later conducted by a havevitrification, been developed in addition to to protect the use cells of against different potential cryoprotective injuries standard ET catheter (Labotect, Straberg, Germany). occurring at subzero temperatures [2]. After ET, suitable remaining embryos were cryopreserved Currently, slow freezing is the most widely used method for cryopreserving human embryos. During this technique, embryos patients, respectively. During those years, all conditions and are exposed to combined controlled cooling rates with the use protocolsusing slow for freezing embryo or culturing virtification were techniqueskept constant. in 322 and 194 of low concentration of cryoprotectants. Alternatively, embryos an equilibration solution, comprising 1.5mol/L 1, 2 propanediol ultra-rapid cooling with minimum volume along with use of high in Ham’sIn the slow F-10 freezing medium, method, supplemented embryos with were 20% first Albuminal-5,incubated in concentrationscan be cryopreserved of cryoprotectants, by vitrification, allowing a technique embryos that tocombines rapidly containing 5% human serum albumin at room temperature for enter a glass-like state [3]. 10min, and then transferred to the freezing solution (1.5mol/L 1, 2 propanediol and 0.5mol/L sucrose in Ham’s F-10 medium), supplemented with 20% Albuminal-5 for an additional 10min. thisSince method the became publication increasingly of the first popular reports among demonstrating embryologists, the Thereafter, three or more embryos were loaded into a plastic feasibility of using vitrification to cryopreserve human embryos, mini-straw and the freezing program was executed. The straw time requirements when compared to slow freezing [4]. The main was then held at -33°C for 30min before it was plunged and concernbecause remains of its significant however, advantagesis the need to with use regardshigh concentrations to cost and stored in liquid nitrogen (LN). of cryoprotective solutions that might lead to osmotic shock, On the day of frozen ET (FET), one of the patient’s straws was which can affect embryo survival [5]. removed from LN (before deciding to thaw the others), exposed to room temperature and then immersed in a water bath at embryos has far been shown by several groups, however the 30°C. The embryos were then transferred from the straw to a currentThe best feasibility available of evidence using vitrification does not allow to cryopreserve for solid conclusion human decreasing concentrations of 1, 2 propanediol in the thawing to be made with regards to the association of this technique with solution (0.5mol/L sucrose and 20% Albuminal-5 in Ham’s F-10) higher pregnancy rate. Therefore, we aimed to review our data 5min before they were transferred to G1 culture media. FET was thenfor 5min conducted. and finally in sucrose-free thawing solution for another survivaland assess and theclinical efficacy pregnancy of slow rates. freezing technique of human embryos and compare it to vitrification, in terms of post-thaw MATERIALS AND METHODS at room temperature in equilibration solution (7.5% ethylene glycolIn vitrification, (EG) and 7.5% suitable dimethyl surplus sulphoxide embryos were (DMSO) first incubated in Ham’s This is a retrospective cohort study conducted at a private F-10 media, supplemented with 20% Albuminal-5) for 5 to In-Vitro Fertilization (IVF) centre in Saudi Arabia. Records from 15min (depending on the time needed for the re-expansion of June 2003 until June 2011 were assessed and the data of 516 cells). After an initial shrinkage and recovery, the embryos were patients who underwent IVF and had surplus luteal day-3 (LD3) embryos that were suitable for cryopreservation was obtained. mol/L sucrose in Ham’s F-10 medium, supplemented with 20% Institutional Ethical Committee approval was obtained. Albuminal-5)transferred into at roomvitrification temperature solution for (15% 60 seconds EG, 15% and DMSO, embryos 0.5 Patients were prepared for oocyte retrieval using the were immediately aspirated and placed at the tip of a Cryotop standard ovarian long stimulation protocol. Down regulation (Vitrolife, Sweden) and then plunged into LN. Three or more was achieved using a gonadotropin releasing hormone (GnRH) embryos were placed on each Cryotop. agonist (Decapeptyl, Ferring, Germany), then ovarian stimulation On the day of FET, one of the patient’s Cryotops were rapidly was initiated using an exogenous recombinant gonadotropin removed from LN (before deciding to thaw the others) and the (Puregon, MSD, Netherlands), with a starting dose of 225 to 300 embryos were exposed to the thawing solution for 50 to 60sec at International Unit (IU). The dose was then adjusted in tandem 37°C and then transferred (at room temperature) into dilution with ovarian follicular development as monitored by serial solution of 0.5 then 0.25mol/L sucrose for 3min each. The serum estradiol and transvaginal ultrasound (TV/US). Oocyte warmed embryos were washed 4-5 times into washing solution retrieval was performed 36 hours (h) after the administration (Ham’s F-10 medium supplemented with 20% Albuminal-5) of 10,000 iu of human chorionic gonadotropin (hCG, Choriomon, before they were transferred to G1 culture media. FET was then IBSA, Switzerland), which was administrated when at least three conducted. follicles reached 17 millimetre (mm) in diameter . Endometrial preparation was achieved using oral estradiol After the oocytes were inseminated, resulting embryos valerate (EV) starting with 4 milligram/day (mg/d) on menstrual underwent standard IVF protocols and were cultured in G1 (Vitrolife, Sweden), supplemented with 10% recombinant human serum albumin for 3 days. On LD3, embryos were scored indays thickness, 1 to 5, then micronized 6mg/d on progesterone MD 6 to 8 and vaginal finally with pessaries 8mg/d were on MD 9 to 12. When the endometrial lining reached at least 8mm

Med J Obstet Gynecol 2(4): 1047 (2014) 2/5 Eskandar et al. (2014) Email: Central commenced at 100mg/d and EV was reduced to 4 g/d. Both were Table 1: Patients’ characteristics. continued until 10 weeks of gestation or the commencement of Slow Freez- Vitrifica- P- OR (95%CI) a spontaneous menstrual period. FET was conducted three days ing tion value after starting progesterone, and a pregnancy test followed by 15 Number of patients 322 0.031 …….. days. If the latter was positive, a TV/US was performed 3 weeks Number of embryos 1607 750194 0.043 …….. produced one intra-uterine gestational sac. 0 .051 later to confirm a clinical pregnancy by the presence of at least Age (years) STATISTICAL ANALYSIS 29.57±4 .68 29.52±5.32 0.91 (95%CI 0.83- Statistical analysis was made with the aid of Microsoft Excel. Duration of embryo storage in months 0.001 0.93) (mean +/-SD) freezing groups were analyzed using chi-square and Fisher’s 4.3 (95%CI Number of frozen 11.08±10.84 6.79±4.72 exactDifferences tests for amongst categorical variables variables, of theand vitrificationthe student’s andt-test slow for 2.68-5.91) embryos 0.001 continuous variables. P<0.05 was considered to be statistically 1.43-2.45) (mean +/-SD) 1.94 (95%CI 7.47±3.48 5.53±2.38 0.237 Blastocyst rate after pregnancy rates between the two groups were presented as 0.001 thawing significant. Comparisons of post-thawed survival and clinical 0.12 0.00±0.00 0.24±0.82 (95%CI0.354- variancesodds ratios and (OR) t-test with for correspondingequality of means. 95% confidence intervals (95%CI). Levene’s test was used for assessing the equality of TableOR: odds 2: ratio; CI: confidence interval. . RESULTS TechniqueEmbryos̓Slow survival freezing after thawingVitrification P-value OR (95%CI) Out of 516 patients, surplus embryos from 322 patients Number of 322 were cryopreserved using the slow freezing technique, while patients

Survival rate 194 0.012 CI 1.55- embryos from 194 patients were cryopreserved by vitrification. 712.45) (95% No significant difference in patients’ age was found between the 69.75±28.06 62.75±31.89 (Mean ±SD) highertwo groups, for the however slow freezing the mean group storage (Table duration 1). was significantly longer and the mean number of frozen embryos was significantly TableSD: standard 3: Clinical deviation; pregnancy OR: oddsrate. ratio; CI: confidence interval The post-thaw survival and clinical pregnancy rates were Technique Slow freezing Vitrification P-value Number of patients 105 37 P=0.012) and (34.8 vs. 21.5, who underwent FET significantly higher for embryos cryopreserved using the slow P=0.002), respectively (Tables 2 and 3). *Clinical pregnancy 34.8 21.5 0.002 freezing technique (69.75 vs. 62.75, rate (percentage) DISCUSSION * At least one intrauterine gestational sac seen on ultrasound FET: frozen embryo transfer for assisted reproduction, as it provides a way to achieve a pregnancy,Cryopreservation without exposingof human embryos the female is a beneficial partner to technique ovarian stimulation or oocyte retrieval procedure, which are time the slow freezing group compared to the vitrification group with consuming, stressful, costly, and are not without complications in the slow freezing method. significant higher post-thaw survival and clinical pregnancy rates [6]. Currently, two cryopreservation methods are used, the Randomized Controlled Trials (RCTs) comparing slow “lengthy” slow freezing method that requires relatively expensive highly toxic levels of cryoprotectants [7]. freezing versus vitrification techniques, in terms of post-thaw equipment, and the vitrification method that involves the use of not.survival All reviewed and clinical studies pregnancy assessed rates the same had primary conflicting outcomes results (Table 4) [13-19]. Some agreed with our results, but others did relatively simpler and less expensive, slow freezing remains to measures we looked at. One study however, added live birth be theAlthough most used vitrification technique is for becoming cryopreserving more attractive human embryos. as it is rate as a secondary outcome measure [14]. Those studies

technique in terms of post-thaw survival rate, whether embryos Over the past few years, several studies with conflicting results wereshowed cryopreserved that vitrification at blastocystappears to orbe cleavagebetter than stage, slow howeverfreezing were published comparing slow freezing with vitrification [8-11]. the metanalysis that included all those studies could not give solid conclusion with regards to the clinical pregnancy rate, and post-thawOur results survival showed and that clinical compared pregnancy to vitrification, rates, althoughthe slow freezing technique was associated with a significantly higher were part of this metanalysis, transferred embryos at different stagesthat is of because development, two of the which most is relevanta factor that studies can [14,19] impact that the embryos̓ storage duration was significantly longer in this group. detrimental effect on its survival or clinical pregnancy rates, clinical pregnancy rate, regardless of the technique used for This may suggest that length of embryos̓ storage time have no which have also been suggested by Riggs et al. [12]. Our group freezing. Additionally, live birth rate, which was assessed by results also revealed that the number of cryopreserved 6 to 8 freezing techniques [14]. Moreover, slow freezing technique was Rama et al., was not significantly different between the two cells of grade I embryos after thawing were highly significant in Med J Obstet Gynecol 2(4): 1047 (2014) 3/5 Eskandar et al. (2014) Email: Central

Table 4:

Summary of studies that compared Slow FreezingOutcome with Vitrification techniques. Results Study Type of study Significance measures Vit SF Li et al.13 RCT PTS & CPR PTS: PTS: NS 88.8% NS CPR: 91.3%CPR: 47.5% 37.5%

Rama et al.14 RCT PTS, CPR & live birth rate PTS: PTS: S 60%

95.3%CPR: CPR: NS 35% 17.4%

Live birth rate: Live birth rate: NS 32.5% 13% Balaban et al.15 RCT PTS & blastulation rate PTS: PTS: S 88.8%

Blastulation94.9% rate: Blastulation rate: S 60.3%

16 Zheng et al. RCT PTS PTS: 49.5%PTS: S 15% Huang et al.17 RCT PTS & CPR PTS: NA NA 94% 77.1%

CPR: NA NA 53.8%

Bernal et al.18 RCT PTS & CPR PTS: PTS: S 76%

CPR:93% CPR: NS 62.5% Kuwayama et al. Prospective CPR for cleavage stage embryos CPR: CPR: NS 54.9% 19 Cohort study & blastocysts cleavage stage embryos cleavage stage embryos 27% 32.1%

blastocyst blastocyst 53% 51% NS

P<0.05; NS: not signi RCT: randomised controlled trial; No.: number; Vit: vitrification; SF: slow freezing; PTS: post-thaw survival rate; CPR: clinical Pregnancy rate; NA: not available; S: statistically significant at number of embryos transferred. ACE added that well-designed in Kuwayama et al. study (32% vs. 27%), however the difference trials with careful follow-up of born children are needed, before associated with a higher clinical pregnancy rate than vitrification an optimal method can be agreed upon. did not reach a statistically significant level [19]. Therefore, it cannot be said with confidence that vitrification is associated Some of those methods (“open” methods) bring eggs and/or Different methods for vitrification have been described. withIn a higher 2011 probability the UK Association of clinical pregnancy of Clinical [9]. Embryologists embryos into direct contact with LN in order to enhance the rate (ACE) held a consensus workshop on Oocyte and Embryo of cooling. It has been suggested that this may increase the risk Cryopreservation. The data supplied suggested that slow of contamination with pathogens,[20] and that slow freezing may have a greater margin of safety [21]. Additionally, some of the stage of embryos’ development (cleavage stage embryos orfreezing blastocysts), and vitrification and the can group lead concluded to similar outcomes, that at the regardless moment that frozen cleavage stage embryos can survive with equivalent there is not enough evidence to recommend either method [20]. numberclinics using of intact the slow cells; freezing as they techniquewere before showed crypreservation data confirming [21]. The report however, stated that care should be taken when The same group reported high rates of survival of fully intact interpreting these data, because of the so many factors involved (other than the cryopreservation technique used) that could have suggesting that FET can potentially be as effective as fresh ET, whenembryos, it comes when the to freezing clinical pregnancyand diluting rate solutions [22]. were Therefore, modified, the the method by which embryos for transfer were selected, and the influenced the results, such as the patient population differences, current best available evidence assessing the relative efficacy of Med J Obstet Gynecol 2(4): 1047 (2014) 4/5 Eskandar et al. (2014) Email: Central

one technique to the other [23]. 10. AbdelHafez FF, Desai N, Abou-Setta AM, Falcone T, Goldfarb J. Slow vitrification versus slow freezing, does not support the shift from Opin Obstet Gynecol. 2009; 21: 270-274. a systematic review and meta-analysis. See comment in PubMed freezing, vitrification and ultra-rapid freezing of human embryos: Another possible explanation for our findings is that our [8].overall Centres experience may needwith toslow go freezing through is a significantly learning curve longer before and 11. CommonsRezazadeh below Valojerdi Reprod M, Biomed Eftekhari-Yazdi Online. 2010; P, 20: Karimian 209-222. L, Hassani theygreater get than comfortable with the relativelyand achieve “new” comparable vitrification results technique with survival, post warming embryo morphology and pregnancy outcomes F, Movaghar B. Vitrification versus slow freezing gives excellent practiced in our centre), use high concentrations of permeating cryoprotectantsvitrification. Additionally such as DMSO.many vitrification Usage of such protocols cryoprotectants, (as the one 12. forRiggs human R, Mayer cleaved J, Dowling-Lacey embryos. J Assist D, Chi Reprod TF, Jones Genet. E, 2009; Oehninger 26: 347-354. S. Does variation in temperatures, as well as variation in the carrier analysis of 11,768 cryopreserved human embryos. Fertil Steril. 2010; system, may have a deleterious effect on the post-thaw embryo storage time influence postthaw survival and pregnancy outcome? An survival rates, which can potentially lower the clinical pregnancy rates as well. 13. 93: 109-115. CONCLUSION Li Y, Chen ZJ, Yang HJ, Zhong WX, Ma SY, Li M. [Comparison of Zhonghuavitrification Fu andChan slow-freezingKe Za Zhi. 2007; of 42: human 753-755. day 3 cleavage stage Slow freezing may yield better results in experienced hands. embryos: post-vitrification development and pregnancy outcomes]. 14. Rama Raju GA, Haranath GB, Krishna KM, Prakash GJ, Madan K. Embryo cryopreservation is an area of active research, justifying the need for larger well-designed controlled trials to strengthen pregnancy rates. Reprod Biomed Online. 2005; 11: 434-437. (or even weaken) this conclusion, and to reliably evaluate Vitrification of human 8-cell embryos, a modified protocol for better the impact of each cryopreservation technique on the clinical 15. Balaban B, Urman B, Ata B, Isiklar A, Larman MG, Hamilton R. A randomized controlled study of human Day 3 embryo cryopreservation pregnancy or even live birth rates, as well as on the potential impact on born children. survival, metabolism and blastocyst formation. Hum Reprod. 2008; by slow freezing or vitrification: vitrification is associated with higher REFERENCES 16. 1. 23: 1976-1982. of the survival of human biopsied embryos after cryopreservation Tissue Bank. 2006; 7: 135-141. Zheng WT, Zhuang GL, Zhou CQ, Fang C, Ou JP, Li T, et al. Comparison Michelmann HW, Nayudu P. Cryopreservation of human embryos. Cell with four different methods using nontransferable embryos. Hum 2. other cryopreservation procedures for oocytes and embryos. Hum Shaw JM, Jones GM. Terminology associated with vitrification and 17. Reprod.Huang CC, 2005; Lee 20:TH, 1615–1618. Chen SU, Chen HH, Cheng TC, Liu CH. Successful pregnancy following blastocyst cryopreservation using super-cooling 3. ReprodEfstratios Update. M. Kolibianakis, 2003; 9: 583-605. Christos A. Venetis and Basil C. Tarlatzis. 18. ultra-rapidBernal DP, Changvitrification. CC, Colturato Hum Reprod. LF, Leef 2005; DM, 20: Kort 122-128. HI, Nagy ZP, et al. Cryopreservation of human embryos by vitrification or slow freezing: Evaluation of blastocyst recuperation, implantation and pregnancy 4. whichTrounson one A,is better?Mohr L. Curr Human Opin pregnancy Obstet Gynecol. following 2009; cryopreservation, 21: 270–274. rates after vitrification/warming or slow freezing/thawing cycles. thawing and transfer of an eight-cell embryo. Nature. 1983; 305: 707- FertilKuwayama Steril. M, 2008; Vajta 90: G, S277–S278.Ieda S, Kato O. Comparison of open and closed 5. 709.Quinn P, Kerin JF. Experience with the cryopreservation of human embryos using the mouse as a model to establish successful 19. potential contamination. Reprod Biomed Online. 2005; 11: 608-614. methods for vitrification of human embryos and the elimination of 20. 6. techniques.Lindheim SR, J In Legro Vitro RS,Fert Morris Embryo RS, Transf. Vijod 1986; MA, Lobo 3: 40-45. RA, Paulson RJ. Altered responses to stress in women undergoing in-vitro fertilization Brison D, Cutting R, Clarke H, Wood M. ACE consensus meeting report: oocyte and embryo cryopreservation Sheffield 17.05.11. Hum Fertil 21. (Camb).Edgar DH, 2012; Bourne 15: 69-74.H, Speirs AL, McBain JC. A quantitative analysis of 7. andSaito recipients H, Ishida of GM, oocyte Kaneko donation. T, Kawachiya Hum Reprod. S, Ohta 1995; N, 10: Takahashi 320-323. T. the impact of cryopreservation on the implantation potential of human

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Cite this article Edris F, Baghdadi S, Fares A, Alasmari W, Dabit S, et al. (2014) Compared to Vitrification, Slow Freezing Technique is Associated With a Higher Post-Thawed Embryos Survival and Clinical Pregnancy Rates. Is This a Myth or a Fact? Med J Obstet Gynecol 2(4): 1047.

Med J Obstet Gynecol 2(4): 1047 (2014) 5/5