Vitrification Cryopreservation Research Articles

An Ultrafast Vitrification Method for Cell Cryopreservation

Ultrafast cooling is the key to successful cell vitrification cryopreservation of lower concentration cryoprotective solution. This research develops a cell cryopreservation methodology which utilizes thin film evaporation and achieves vitrification of relatively low concentration cryoprotectant with an ultrafast cooling rate. Experimental results show that the average cooling rate of dimethylsulfoxide (DMSO) cryoprotective solution reaches 150,000 °C/min in a temperature range from 10 °C to −180 °C. The ultrafast cooling rate can remarkably improve the vitrification tendencies of the cryoprotective solution. This methodology opens the possibility for more successful cell vitrification cryopreservation.

Evaporation heat transfer of liquid nitrogen on microstructured surface at high superheat level

Liquid nitrogen, as a coolant, is generally applied in cell vitrification cryopreservation. It takes heat from the carrier with cell samples through its violent evaporation on the carrier surface. As a result, the temperature of the carrier plunges dramatically. This article focuses on the unsteady evaporation heat transfer characteristics of liquid nitrogen on a microstructured surface etched into the frozen carrier surface at a high superheat level. The heat flux and evaporation heat transfer coefficient of liquid nitrogen were investigated using a lumped capacitance method. The experimental results showed that the cooling rate of the thin film evaporation on the microstructured surface is obviously higher than that of pool boiling, which is currently being used for cell cryopreservation. The heat flux and the evaporation heat transfer coefficient work together to present a parabolic trend with the superheat decreasing during this heat transfer process. Besides, the microstructure of the surface has an important effect on the evaporation heat transfer of liquid nitrogen. The larger the thin film evaporation zone is, the higher the heat transfer coefficient is. The current investigation results in a cell cryopreservation method through vitrification with relatively low concentrations of cryoprotectants.

Modeling and experimental studies of enhanced cooling by medical gauze for cell cryopreservation by vitrification

Vitrification is considered as an important alternative approach to traditional slow freezing method for cryopreservation of cells. A typical cell vitrification procedure involves a non-equilibrium cooling process commonly accomplished in liquid nitrogen, while in which film boiling is believed to greatly hinder heat transfer surrounding the sample, resulting in incomplete vitrification or a much higher critical concentration. In this study, we developed a simple while effective approach, wrapping traditional French-type straw with medical gauze, to greatly enhance convective heat transfer during cooling by suppress film boiling. We further established a coupled heat transfer model for cooling and warming of cell suspensions to investigate the inherent thermodynamic mechanism in this approach. The model describes both the macroscale thermal distributions in extracellular solution and the microscale ice crystallization inside the cells. The simulation indicated that straws wrapped with medical gauze would increase cell survival subject to vitrification cryopreservation by significantly increasing the cooling rate to inhibit intracellular ice formation (IIF). Our experiments on human umbilical vein endothelial cells (HUVECs) further confirmed the predictions in that the cell survival rate was significantly increased by wrapping straws with medical gauze.

Integration of Cryopreservation and Tissue Culture for Germplasm Conservation and Propagation of Rosa pomifera ‘Karpatia’

Cryopreservation is an useful technique for long-term conservation that requires minimal space and maintenance. Germplasm protection of Rosa is important to preserve genetic diversity, to store material for breeding and to expand new research. This study was conducted to develop a droplet vitrification cryopreservation and micropropagation of Rosa pomifera cv. ‘Karpatia’, whose large hypanthia are characterized by remarkable pro-health properties. Culture in vitro was stabilized and shoot tips collected from dormant buds served as initial explants. The multiplication of shoots was carried out on MS medium containing benzyladenine. For the droplet vitrification cryopreservation, shoot tips from in vitro cultures were used: small with exposed meristem, and large with a meristem covered with leaves, as well as shoot tips from in situ plants, which were collected in winter. Treatment time with plant vitrification solution (PVS2) was also tested (10-30 minutes). From in vitro culture, 32-41% small explants with exposed meristem survived, but they regenerated at a very low level. The best cryostorage results were obtained for shoot tips from dormant buds and a 20-minute PVS2 treatment: the survival was 84% and regeneration 72%. During the post-freezing regeneration multiplication index was 2.4 shoots per one multiplication cycle, after cryopreservation and in the control. On half MS medium without growth regulators, 97-99% of shoots rooted, and all rooted plants have adapted to ex vitro conditions and were planted into the soil. Biometric analyses during shoot multiplication, rooting and acclimatization stages did not reveal any changes compared to the non-cryopreserved samples.

Chapter 18 Vitrification: A Reliable Method for Cryopreservation of Animal Embryos.

The impact of cryopreservation in assisted reproduction is increasingly appreciated. Cells, oocytes, and embryos preserved under ultralow temperature can endure storage for indefinite time with almost no alteration in their metabolic and genetic components. Advances in cryopreservation and its applications to preserve cells, gametes, and embryos offer opportunities in conservation of biodiversity and dissemination of elite livestock germplasm. This chapter describes the vitrification for cryopreservation of livestock embryos.

41 THE EFFECT OF ULTRARAPID VITRIFICATION FOR SHEEP OOCYTE VIABILITY

Advances in reproduction technologies, such as in vitro maturation, IVF, and in vitro culture, have stimulated research for efficient cryopreservation techniques for mammalian oocytes. It is well known that the oocyte is the largest cell of an animal’s body and as such, is full of water and, in many species, fat, making it difficult to cryopreserve. The objective of this work was to study the effect of vitrification for cryopreservation of the metaphase II plate (MPII) of sheep oocytes. In our experiment, we used the Vit-Master™ (MTG, Bruckberg, Germany). Ovaries from 19 ewes of Kazakh Arkharo-Merino breed were acquired after slaughter and maintained at 37°C in TCM-199. The maturation medium was TCM-199, containing 1 mM of glutamine, 10% fetal bovine serum, 5 μg mL−1 FSH, 5 μg mL−1 LH, 1 μg mL−1 oestradiol, 0.3 mM sodium pyruvate, and 100 mM cysteamine. The oocytes were incubated in 400 μL of medium in 4-well dishes covered with mineral oil. The IVM conditions were 5% CO2 in humidified air at 39°C for 24 h. Then, oocytes were placed for 10 min in medium with Hoechst 33342 (3 μg mL−1) and cytochalasin B (7 μg mL−1) to facilitate enucleation of the MPII with a minimum volume of ooplasm. The MPII plates were divided into 2 groups: the vitrification group was exposed to vitrification media containing 1.12 M ethylene glycol (ET) + 0.87 M ME2SO for 5 min and was exposed in vitrification media containing 2.24 M ET + 1.75 M ME2SO for 5 min, and then in vitrification solution containing 4.48 M ET + 40% ME2SO + 0.25 M sucrose for 30 s. Oocytes were loaded into a cryoloop and using negative pressure of liquid nitrogen in the chamber for freezing with the VIT-Master. Oocytes were thawed in a 25°C water bath and then placed in TCM-199 at 20% fetal bovine serum. After 15 min of incubation, the oocytes were activated for extrusion of the second polar body in 1 mg mL−1 Ca ionophore for 5 min and washed for 5 min followed by 4 h in 6-DMAP (0.12 mM) + cycloheximide (0.6 μg mL−1). After activation, the MPII were washed and cultured for 20 h. The control group received the same treatment but were not vitrified. Differences between the experimental groups were tested using Chi-squared test. Our research showed that expulsion of the second polar body after activation was observed in more than 59.7% of the MPII that were not vitrified (control group), whereas 37.7% of vitrified plates had expulsion of polar bodies (P < 0.05). These preliminary studies showed that it is possible to vitrify MPII plates. On the other hand, the drastic reduction of the volume of the sheep oocytes might make cryopreservation possible with greater efficiency.

Ascorbate Peroxidase Activity of Aranda Broga Blue Orchid Protocorm-like Bodies (PLBs) In Response to PVS2 Cryopreservation Method.

Throughout the cryopreservation process, plants were exposed to a series of abiotic stresses such as desiccation and osmotic pressure due to highly concentrated vitrification solution. Abiotic stress stimulates the production of reactive oxygen species (ROS) which include hydrogen peroxide, superoxide radicals, and singlet oxygen. Higher production of ROS may lead to oxidative stress which contributes to the major injuries in cryopreserved explants. Antioxidant enzymes in plant such as ascorbate peroxidase (APX) can protect plants from cell damage by scavenging the free radicals. This study was determined based on APX enzyme activity of Aranda Broga Blue orchid's protocorm-like bodies (PLBs) in response to PVS2 (Plant Vitrification Solution 2) cryopreservation treatments at different stages. PLBs that were precultured at 0.25 M sucrose for 3 days were subjected to vitrification cryopreservation method. Results obtained showed that the highest APX activity was achieved at PVS2 cryoprotectant treatment prior liquid nitrogen (LN) storage. This phenomenon indicating that accumulation of osmotic and dehydrating stress throughout the cryopreservation treatment resulted in oxidative burst which in turn leads to higher APX activity in order to control the excess production of ROS. To conclude, PVS2 treatment was revealed as the most detrimental step throughout cryopreservation treatment. Thus, this research also suggested that exogenous antioxidant such as ascorbic acid can be added throughout cryopreservation procedure especially at PVS2 treatment in the future experiments to aid in regrowth of cryopreserved explants by reducing oxidative stress.

The effect of high electromagnetic field heated magnetic nanoparticles on biomaterial vitrification cryopreservation

The effect of high electromagnetic field heated magnetic nanoparticles on biomaterial vitrification cryopreservation

The impact of physiological oxygen during culture, and vitrification for cryopreservation, on the outcome of extended culture in human IVF

Extended culture has facilitated the move to single blastocyst transfer, resulting in significant increases in implantation and live birth rate, while concomitantly reducing fetal loss during pregnancy. However, concerns have been raised regarding subsequent neo-natal outcomes following extended culture. Analysis of the literature reveals differences in outcomes according to geographical region and between individual clinics. A common factor amongst reports of potentially adverse outcomes following blastocyst transfer appears to be that atmospheric (~20%) oxygen was typically employed for embryo culture. Clinics and countries utilizing physiological concentrations of oxygen (~5%) have not reported adverse perinatal outcomes with blastocyst transfer. Atmospheric oxygen imposes significant negative effects upon the embryo's molecular and cellular physiology, and further it increases the sensitivity of the preimplantation embryo to other stressors in the laboratory. With the recent adoption of vitrification for blastocyst cryopreservation, cumulative pregnancy rates per cycle with extended culture will increase significantly. Consequently, rather than perceiving extended culture as a potentially negative procedure, it is concluded that neo-natal data need to be interpreted in light of the conditions used to culture and cryopreserve blastocysts, and that furthermore a policy of embryo culture using 20% oxygen can no longer be justified.

Field performance evaluation and genetic integrity assessment in Argyranthemum ‘Yellow Empire’ plants recovered from cryopreserved shoot tips

Field performance evaluation and genetic integrity assessment were conducted in Argyranthemum plants derived from cryopreserved shoot tips. Some variations in root formation and vegetative growth were found in the plants following cryopreservation, but morphologies of the leaves and flowers, and color, number and size of the flowers remained unchanged in the plants recovered from cryopreservation, compared with the control. Assessments of genetic integrity by the two molecular markers: inter simple sequence repeat (ISSR) and amplified fragment length polymorphism (AFLP) did not detect any polymorphic bands across the plants tested following cryopreservation. These data indicate that cryopreservation reduces, to a certain degree, root formation and vegetative growth, but it does not alter morphologies of leaves and flowers, may not cause any genetic alternations, and has no adverse effects on quantity and quality of the flowers. Therefore, the droplet vitrification cryopreservation can be considered promising for long-term preservation of Argyranthemum germplasm.

Phasing in and out of the FREEZE-ALL mentality: was Mother Nature right after all?

Phasing in and out of the FREEZE-ALL mentality: was Mother Nature right after all?

56 THE EFFECT OF VITRIFICATION FOR SHEEP OOCYTES VIABILITY

Advances in reproduction technologies, such as in vitro maturation, IVF, and in vitro culture, stimulated research for efficient cryopreservation techniques for mammalian oocytes. It is well known that the oocyte is the largest cell of an animal's body and as such, is full of water and, in many species, fat, making it difficult to cryopreserve. The objective of this work was to study the effect of vitrification for cryopreservation of the metaphase II plate (MPII) of sheep oocytes. Ovaries from 20 ewes of Kazakh Arkharo-Merino breed were acquired after slaughter and maintained at 37°C in TCM-199. The maturation medium was TCM-199, containing 1 mM of glutamine, 10% FBS, 5 μg mL–1 FSH, 5 μg mL–1 LH, 1 μg mL–1 oestradiol, 0.3 mM sodium pyruvate, and 100 mM cysteamine. The oocytes were incubated in 400 μL of medium in 4-well dishes covered with mineral oil. The IVM conditions were 5% CO2 in humidified air at 39°C for 24 h. Then they were placed for 10 min in a media with Hoechst 33342 (3 μg mL–1) and cytochalasin B (7 μg mL–1) to facilitate the enucleation of the MPII with a minimum volume of ooplasm. The MPII plates were divided into 2 groups: the vitrification group was exposed to vitrification media containing 1.12 M ethylene glycol (ET) + 0.87 M ME2SO for 5 min and was exposed in vitrification media containing 2.24 M ET + 1.75 M ME2SO for 5 min, and then in vitrification solution containing 4.48 M ET + 40% ME2SO + 0.25 M sucrose for 30 s. Oocytes were loaded into cryoloop and plunged into liquid nitrogen (LN2). Oocytes were thawed in a 25°C water bath and then placed in TCM-199 at 20% fetal bovine serum. After 15 min of incubation the oocytes were activated for extrusion of the second polar body in 1 mg mL–1 Ca ionophore for 5 min and washed for 5 min followed by 4 h in 6-DMAP (0.12 mM) + cycloheximide (0.6 μg mL–1). After activation the MPII were washed and cultured for 20 h. The control group received the same treatment, but they were not vitrified. Differences between the experimental groups were tested using Chi-squared test. Our research showed the expulsion of the second polar body after activation was observed in more than 62.2% of the MPII that were not vitrified (control group), whereas 40.5% of vitrified plates had expulsion of polar bodies (P < 0.05). These preliminary studies showed that it is possible to vitrify MPII plates. On the other hand, the drastic reduction of the volume of the sheep oocytes might make cryopreservation possible with greater efficiency.

Ongoing and cumulative pregnancy rate after cleavage-stage versus blastocyst-stage embryo transfer using vitrification for cryopreservation: impact of age on the results.

To determine if blastocyst transfer increases the ongoing and cumulative pregnancy rates, compared with day 3 embryo transfer, in women of all ages when at least 4 zygotes are obtained. Prospective study including patients undergoing a first IVF/ICSI treatment and assigned to cleavage stage (n = 46) or blastocyst (n = 58) embryo transfer. Supernumerary embryos were vitrified and patients failing to achieve an ongoing pregnancy after fresh embryo transfer would go through cryopreserved cycles. The main outcome measure was the ongoing pregnancy rate after the fresh IVF/ICSI transfer and the cumulative ongoing pregnancy rate. Results were also analyzed according to age (under 35 and 35 or older). A majority of patients (96.6 %) had a blastocyst transfer when at least 4 zygotes were obtained. The ongoing pregnancy rate was significantly higher in the day-5 group compared with the day-3 group (43.1 % vs. 24 %, p = 0.041). The cumulative ongoing pregnancy rate was higher (but not significantly) with blastocyst than with cleavage stage embryos (56.8 % vs. 43.4 %, p = 0.174). When analysed by age, patients 35 or older showed significantly higher ongoing pregnancy rate (48.4 % vs. 19.3 %, p = 0.016) and cumulative ongoing pregnancy rate (58 % vs. 25.8 %, p = 0.01) in the day-5 group compared to the day-3 group, while no such differences were observed in women under 35. Blastocyst transfer can be suggested whenever there are at least 4 zygotes. While there are no differences in women under 35, the benefit of this option over cleavage stage transfer could be significant in women 35 or older.

Phenotypic and molecular studies for genetic stability assessment of cryopreserved banana meristems derived from field and in vitro explant sources

Explants used for cryopreservation of banana (Musa L. spp.) are mainly sourced from tissue culture. Here, we demonstrate the successful use of sucker meristems (SM) obtained from field-raised plants for cryopreservation of Indian Musa ABB cv. ‘Karpura Chakkarakeli’. In addition, the genetic stability of plants recovered from cryopreserved and regenerated meristems after hardening and transfer to field conditions was studied using 11 phenotypic (biometric) characters and 21 simple sequence repeat (SSR) markers. The regenerative potential of cryopreserved SM was compared with two types of routinely employed explants of banana germplasm: in vitro-raised single-shoot meristems (IVM) and proliferating meristems (PM). The regeneration frequency of SM was high (60.0 ± 11.5%) and statistically comparable to PM (68.3 ± 4.4%) and IVM (55.6 ± 11.1%) after using the droplet vitrification cryopreservation technique. The total time required for cryopreserving plants from SM (∼2 mo) was substantially less than that for PM (14 mo) and IVM (8 mo). The SSR profiles of plants recovered from cryopreserved PM, IVM, and SM and compared with control plants had a similarity coefficient of 0.92. Data on phenotypic traits revealed that cryopreserved plants were statistically comparable to the mother plants raised from suckers for all important growth and yield parameters. This study broadens the possibilities to cryopreserve Musa germplasm, by applying the droplet vitrification method to a new type of explant, the SM. The results presented in this paper show that Musa meristems can be effectively cryopreserved for storage and regeneration of true-to-type plants.

136 BLASTOCOELE COLLAPSE IMPROVES POST-THAW SURVIVAL OF SLOW FROZEN AND VITRIFIED IN VITRO-PRODUCED BOVINE EMBRYOS

Slow-freeze cryopreservation of in vitro-produced bovine embryos often results in poor post-thaw embryo viability. Results with vitrification tend to be better but do not always produce consistent results for a variety of reasons. Experiments with human and equine embryos have demonstrated improved post-thaw survival when blastocoele fluid is removed before vitrification. We hypothesised that removing blastocoele fluid before vitrification or slow-freeze cryopreservation would improve post-thaw survival of in vitro-produced bovine embryos. Three replicates of embryos were generated from abattoir-derived ovaries using standard techniques. Seven days post-fertilization, embryos were evaluated for quality, and grade 1 or 2 embryos were allocated to 1 of 4 treatments: slow-freeze control (n = 103); slow-freeze collapsed (n = 106); vitrified control (n = 117); vitrified collapsed (n = 99). Blastocoele fluid was removed by aspiration with a pipette advanced to the centre of the blastocoele while the embryos were stabilised with a holding pipette. After collapse, embryos were immediately slow frozen in commercial medium [1.5 M ethylene glycol (EG) + 0.1 M sucrose, Bioniche] in 0.25-mL straws or vitrified in 2 steps: 3 min in 1.5 M EG, 30 s in 7 M EG + 0.6 M galactose + 18% Ficoll in open-pulled straws. After thawing or warming, embryos were cultured in chemically defined medium-2 + 5% FCS in 5% CO2, 5% O2, 90% N2 at 38.5°C. Embryos were evaluated for survival based on re-expansion of the blastocoele after 24 h in culture. Data were analysed by GLIMMIX (SAS Institute Inc., Cary, NC, USA). There were significant replicate effects but there were no significant differences in survival between embryos that were vitrified or slow frozen. Approximately twice as many collapsed embryos survived compared to intact control embryos regardless of whether the embryos were vitrified or slow frozen (collapsed: 50.5 ± 4.1%, control: 26.3 ± 3.5%; P < 0.001). Advanced blastocysts (fully expanded and hatched blastocysts) survived better than early blastocysts (pre-expansion blastocysts and expanding blastocysts, 45.9% ± 3.9 v. 30.0% ± 4.4, respectively; P < 0.01). The only significant interaction (P < 0.05) was that vitrification was superior to freezing for early blastocysts (39.5 ± 5.9% v. 22.1 ± 5.0% survival) but not advanced blastocysts (44.4 ± 5.3% v. 47.4 ± 5.8% survival). Removal of blastocoele fluid before cryopreservation of in vitro-produced bovine embryos may be a useful technique for improving post-thaw survival regardless of cryopreservation method or blastocyst stage.

Cryopreservation of human oocytes, zygotes, embryos and blastocysts: A comparison study between slow freezing and ultra rapid (vitrification) methods

Abstract Preservation of female genetics is currently done primarily by means of oocyte and embryo cryopreservation. The field has seen much progress during its four-decade history, progress driven predominantly by research in humans. It can also be done by preservation of ovarian tissue or entire ovary for transplantation, followed by oocyte harvesting or natural fertilization. Two basic cryopreservation techniques rule the field, slow-rate freezing, the first to be developed and vitrification which in recent years, has gained a foothold. The slow-rate freezing method previously reported had low survival and pregnancy rates, along with the high cost of cryopreservation. Although there are some recent data indicating better survival rates, cryopreservation by the slow freezing method has started to discontinue. Vitrification of human embryos, especially at early stages, became a more popular alternative to the slow rate freezing method due to reported comparable clinical and laboratory outcomes. In addition, vitrification is relatively simple, requires no expensive programmable freezing equipment, and uses a small amount of liquid nitrogen for freezing. Moreover, oocyte cryopreservation using vitrification has been proposed as a solution to maintain women’s fertility by serving and freezing their oocytes at the optimal time. The aim of this research is to compare slow freezing and vitrification in cryopreservation of oocytes, zygotes, embryos and blastocysts during the last twelve years. Therefore, due to a lot of controversies in this regard, we tried to achieve an exact idea about the subject and the best technique used.

Stainless steel tube-based cell cryopreservation containers

This study focused on increasing the freezing rate in cell vitrification cryopreservation by using a cryopreservation container possessing rigid mechanical properties and high heat-transfer efficiency. Applying a fast freezing rate in vitrification cryopreservation causes a rapid temperature change in the cryopreservation container and has a substantial impact on mechanical properties; therefore, a highly rigid cryopreservation container that possesses a fast freezing rate must be developed. To produce a highly rigid cryopreservation container possessing superior heat transfer efficiency, this study applies an electrochemical machining (ECM) method to an ANSI 316L stainless steel tube to treat the surface material by polishing and roughening, thereby increasing the freezing rate and reducing the probability of ice crystal formation. The results indicated that the ECM method provided high-quality surface treatment of the stainless steel tube. This method can reduce internal surface roughness in the stainless steel tube, thereby reducing the probability of ice crystal formation, and increase external surface roughness, consequently raising convection heat-transfer efficiency. In addition, by thinning the stainless steel tube, this method reduces heat capacity and thermal resistance, thereby increasing the freezing rate. The freezing rate (3399±197°C/min) of a stainless steel tube after interior and exterior polishing and exterior etching by applying ECM compared with the freezing rate (1818±54°C/min) of an original stainless steel tube was increased by 87%, which also exceeds the freezing rate (2015±49°C/min) of an original quartz tube that has a 20% lower heat capacity. However, the results indicated that increasing heat-transferring surface areas and reducing heat capacities cannot effectively increase the freezing rate of a stainless steel tube if only one method is applied; instead, both techniques must be implemented concurrently to improve the freezing rate.

Theoretical investigations of a novel microfluidic cooling/warming system for cell vitrification cryopreservation

Ultra-fast cooling and rewarming are essential to vitrification cryopreservation. In this work the existing cooling/warming approaches are reviewed and then a novel microfluidic system is proposed. The main concerns of the design includes: (a) enhancing the external heat transfer coefficients by utilizing micro-channel array on the chip surface; (b) increasing the sample surface versus its volume by holding the sample inside the system as an ultra-thin film. By alternatively using liquid nitrogen or warm water as its working fluid, the system can be used for both cooling and warming. To assess its application for achieving ultra-fast cooling/warming rates, the magnitudes of external heat transfer coefficients (h) during cooling and warming are theoretically investigated, and then the transient temperature distribution, the cooling/warming rates and the vitrification/devitrification tendencies of sample solution are numerically studied. It is concluded that an ultra-high h (>104W/m2K) can be obtained by the microfluidic system in both cooling and warming processes, which efficiently improves the cooling/warming rates and then decreases the degree of ice formation. Moreover, the system demonstrates good feasibility in the capacity of sample volume. Therefore, the microfluidic method should be a promising approach for improving vitrification cryopreservation.

Dimethylsulfoxide vs. 1, 2 Propandiol as Cryoprotective Additives during Vitrification in Cryopreservation of Human Embryos

Dimethylsulfoxide vs. 1, 2 Propandiol as Cryoprotective Additives during Vitrification in Cryopreservation of Human Embryos

The fresh vs. all vitrified-thawed embryos transfer of PCOS patients having ovarian hyperstimulation syndrome show similar clinical outcome

Women having PCOS are at high risk of developing ovarian hyperstimulation syndrome undergoing IVF. Many efforts have been progressing to reduce the incidence of OHSS but it couldn't completely get over it. Recently vitrification in cryopreservation has improved the survival rate of embryos. So delaying embryo transfer for the next cycle could give them a better chance to be pregnant.