The Effects of Two Different Freezing Method on Survival and Development of Pronuclear Stage Human Embryos Different Freezing on Survival and Development of Pronuclear Stage

This [2]. A and Abstract : Nowadays, cryopreservation of human embryos has become a required part of IVF programs. The purpose of this study was to compare the recovery, survival, cleavage and blastocyst development rate of pronuclear stage human embryos, after slow freezing and vitrification methods. Material and Methods : Human 2PN stage embryos were divided randomly into four groups. In the 1 M (control) group 29 embryos included without any exposure. In the sham or 2 nd group 15 embryos were considered to survey for cryoprotectant toxicity. Embryos in the 3 rd and 4 th group freezed by vitrification and slow freezing methods respectively. Embryos in 3 rd group (n=36), were vitrified using a new vitrification solution. Survived embryos were cultured, incubated and evaluated for 5 days post-thawing. In 4 group, 2PN embryos (n=41) were freezed in propanediol (PROH) and sucrose 16-18 hours pos-ICSI using a programmed freezing instrument (Planner Series III) and plunged into liquid nitrogen. Results : Our data showed that survival rate in 1 M (control) group was significantly (P<0.05) more than other groups, meanwhile cleavage rate in the control group is significantly more than only slow freezing (4th) group (96.55% vs 76=19%). The blastocyst formation rates in the 3 rd and 4 th groups are significantly lower than control group (59.09% and 42.85% vs 65.51%). Comparison of cleavage and blastocyst formation rate showed a significant difference between 3 rd and 4 th groups. The survival rate has a significant difference between groups (100%, 93.33%, 70.96% and 65.62% respectively) (P<0.05). Discussion : The pronuclear stage embryos from ICSI could choosen for cryopreservation because the developmental capacity of the embryos, after thawing, can easily be ascertained and the predominance of study has shown that human embryos survive and implant at higher rates, when frozen in 2PN. Conclusion : Both procedures had nearly similar effect on 2PN human embryos, but the vitrification method is better because of its simplicity, quickness and cost effectiveness.

crupreservation is vitrification [3,4]. The high concentration of cryoprotectant in the vitrification solution is so that when immersed into liquid nitrogen the solutions form a glass entity; this prevents the formation of extra-and /or intracellular ice crystals during cooling and thawing [2]. All known penetrating cryoprotectants are toxic at high concentrations [1,[5][6][7][8]. Previous studies have indicated that addition of a nonpermeating agent, such as sucrose or polymer (for e.g. ficoll or dextran) to the mixture allows both intra-and extracellular vitrification at less toxic concentrations of permeating cryoprotectant agcents and improves survival rate [1,[9][10][11][12].
It was shown that solutions containing 35% ficoll and 25% ethylene glycol used in vitrification cause high rate of blastocyst from 2-cell mouse embryos [1]. In cru preservation the embryos were chosen at 2PN stage because: i.
it permits the study of a uniform population of embryos at interphase of mitotic division. without any fragmnts which are known to influence cryopreservation [2,13], ii. the post-thawing developmental capacity of the embryos, can easily be ascertained [2,14]and iii. most of the study has shown that human embryos, when frozen in 2PN, survive and implant at higher rates in comparison with the cleavage stage [4,[15][16][17]. Although a variety of more effective slow and rapid Cooling procedures have been developed for cow and mouse embryos, these established protocols are not necessarily effective for other species embryos [1]. It was therefore decided to examine the effectiveness of this new solution on the recovery, survival, cleavage and blastocyst formation rate of 2PN embryos postvitrification and comparing. The aim of this study was to introduce a cryopreserved method which not only had fewer negative effects on survival, cleavage and development but also was simpler, faster and more cost effective.

Vitrification Method
The vitrification of 2PN embryos performed according previously described method [1]. The sibling 2PN embryos of same patients were placed in PBS medium (at room temperature) for

Cryopreservation Protocol
The supernumerary 2PN embryos of each patient were cryopreserved if their tow pronuclei aligned at the equatorial plate, the cytoplasm was clear and had no fragmentation [14,15].
In order to avoid the intervening factors, sibling embryos of each patient were divided randomly into four groups. The embryos in the 1st (control) group (n=29) included without any exposure. In the sham 2 nd group (n=15) embryos were considered to survey for cryoprotectant toxicity. Embryos in the 3 rd (n=36) and 4th The temperature was lowered to -7 °C at a rate of -2 °C/min.

Statistical Analysis
Statistical analysis was done by using X² test. P values less than 0.05 were considered as significant.

Results
In this study a total number of 121 Pronuclear stage embryos were used and divided into control or non-frozen, (n=29), sham (n=15), 1st experimental (vitrification, n=36) and 2nd experimental (slow-freezing, n=41) groups (Table 1). In control group, the mean percent of survival, cleavage and developmental rate were 100, 96.55 and 65.51 percent, respectively. Assessment of 2PN embryos that were only exposed to vitrification solutions (sham group) showed that the survival, cleavage, and development to blastocyst had no significant difference (P>0.05) between groups ( Table 1).
The number of post-thawing recovered embryos in vitrification 31 (86.11%) and slow freezing 32(78.04%) groups have significant difference (P<0.05). The survival, cleavage and blastocyst formation rate in the 3 rd and 4 th expermental groups were shown in Table 1.
Comparison of survival rate between control (1 st ) and slow freezed (4 th ) groups showed that there was a significant difference between groups, similarly the difference between cleavage and blastocyst formation rate in these groups was significant. Comparison of survival rate between non-frozen vitrified (2 nd or sham) and vitrified (3 rd ) groups showed that the difference between them was significant, but the cleavage and developmental rate was not significant (P<0.05) ( Table 1). This study showed that there has a non-significant difference for survival rate between vitrification (3 rd ) and slow freezing (4 th ) groups while the cleavage and development rate have significant difference (P<0.05) ( Table 1).  [3,24]. Additionally, vitrified cells are more sensitive to hypotonic stress than fresh cells in post-warming duration [25]. To reduce the hypoosmotic stress, non-permeating compounds are used as osmotic buffers during removal of permeating cryoprotectant from inside the cells [25]. Breaking of the vitrification solution (during cooling) is another reason for damaging the embryos and reducing the embryos survival rate finally [26], which is in agreement with Liebermann et al, that decrease in survival rate after vitrification is itself a problem [27].
The non-significant difference of cleavage and BDR between  [2,23,28,29] which is in concordant with the-our finding in the present study.
Studies of Kuleshova and, Abbeel showed that during rapid Cooling procedures, number of surviving embryos with a sustained capacity for further cleavage is depended on an optimal exposure time for cryoprotectant solution before freezing.
This finding showed that permeating of the penetrating cryoprotectant is very important [1,2]. For human pronuclear stage embryos, neither the optimal concentration of cryoprotective agent,

Conflict of interest
There is no conflict of interest between authors and none of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper.