Overview of Oocyte Freezing, Embryo Freezing and Sperm Freezing Processes

Abstract:

Advanced Fertility Treatment Options: Dimension of Cryopreservation. Overview of Oocyte Freezing, Embryo Freezing and Sperm Freezing Processes. 

Cryopreservation is nowadays an integrated part of Assisted Reproductive Technologies. Undisputedly, it represents an attractive option to the range of Infertility Treatments available at present. The article focuses on researching the peculiarities of Oocyte Cryopreservation, Embryo Cryopreservation and Sperm Cryopreservation technologies. The purpose of the present article was to evaluate the effect of Cryopreservation on oocytes, embryos and spermatozoa and find out the most important factors determining oocytes, embryos and spermatozoa viability. Having observed the numerous scientific articles and analytical overviews concerning this question, we revealed three main integrative phases of Cryopreservation process: 

1. What stages does Oocyte/Embryo/Sperm Cryopreservation process include; 
2. The essence of Cryopreservation procedures; 
3. Factors, which can affect oocyte/embryo/sperm survival rates after Cryopreservation.

Introduction

Dimension of Cryopreservation is highly debated theme among the representatives of scientific elite, respected experts and distinguished researchers. There are many contradictions about the Cryopreservation methods, techniques, technologies, procedures and decisions regarding Oocyte Freezing, Embryo Freezing and Sperm Freezing.

Undisputedly, Cryopreservation represents an attractive option to the range of Infertility Treatments available at present. There are two important issues, however, remain to be resolved before the role of Cryopreservation Technology in current practice can be fully elucidated. Firstly, it would be preferable if continuing scientific research could determine whether modifications to all the Cryopreservation methods could improve outcome. Secondly, a tendency to adopt modernized Cryopreservation Techniques widely only after they show high–quality results.

Technically, the possibility of offering highly efficient oocyte/embryo and Sperm Cryopreservation [Vitrification] needs not only modifications for system Vitrification and usage of high security closed Vitrification devices, but also inclusive scientific investigation of the most delicate methods of whole Cryopreservation procedure and very accurate choice of the Cryopreservation Technologies, considering individual morphological features and biophysical parameters [in every single case].

The respective importance of these most common criteria (oocyte survival, blastomere survival, sperm survival and resumption of mitosis, spermatozoa’s ability to recognize the Zona Pellucida and ability to bind to it (sperm–oocyte interaction: the initial binding of acrosome–intact spermatozoa occurs through terminal α– and β–galactose of the ZPC [60]; other zona proteins are also involved in this primary binding [66], the completion of meiotic maturation with the extrusion of the second Polar Body, the metabolic activation of the previously quiescent oocyte, the decondensation of the sperm nucleus and the maternal chromosomes into the male and female pronuclei respectively, and the cytoplasmic migrations of the pronuclei, which bring them into apposition), that may help fertility experts to select the best oocyte/embryo to transfer after thawing, remains a matter of debate or to select the best semen samples for In Vitro Fertilization (IVF) or Intracytoplasmic Sperm Injection (ICSI) Treatment cycles. It is therefore essential to use the right indications to allow the usage of the best oocytes and the best semen samples for In Vitro Fertilization (IVF) or Intracytoplasmic Sperm Injection (ICSI) Treatment cycles and the transfer of embryos with the best prognosis in order to enhance the possibility of pregnancy.

The recently established possibility of effectively cryopreserving functional oocytes through Vitrification promises to revolutionize In–Vitro Fertilization (IVF) practice. The inefficiency of conventional slow–freezing techniques has for decades prevented the widespread implementation of Oocyte Cryopreservation in clinical practice. The introduction of Oocyte Vitrification significantly advanced the outcome of Oocyte Cryopreservation.

From the current data, it appears that Vitrification is more efficient than slow freezing. Vitrification is achieved by combining a high concentration of cryoprotectants with high cooling and warming rates. Unlike slow freezing, Vitrification results in the complete elimination of ice crystal formation avoiding the main cause of Cryopreservation injury. These high cooling and warming rates are usually achieved by the use of open systems in which the samples are put into direct contact with Liquid Nitrogen during Vitrification, allowing possible contamination.

Cryopreservation of supernumerary embryos is nowadays a well–accepted procedure in Assisted Reproduction programmes. Cryostorage of embryos has offered a valuable complement to this strategy and aims to reduce the number of embryos transferred per procedure. It thus provides many clinical benefits, including the possibility of increasing the cumulative pregnancy rate from a given Treatment cycle while reducing the risk of multiple pregnancy and avoiding the risk of Ovarian Hyperstimulation Syndrome.

However, it has been shown that not all embryos survive the Cryopreservation procedure. Various factors have been reported to influence the outcome of frozen–thawed embryos including: embryo cleavage stage, prefreeze morphological appearance, hormone supplementation during the frozen–thawed Embryo Transfer cycle; Ovarian Stimulation procedure used before oocyte collection and the outcome of the fresh Embryo Transfer cycle. After thawing embryo cells may survive totally, partially or not at all. Cryopreservation damage to embryos can be induced by several factors, including intracellular ice formation, solution effects, osmotic effects, or physical damage by growing ice crystals at the advancing ice front. When cryopreserved embryos are recovered from Liquid Nitrogen, embryos with damaged blastomeres and/or with a cracked Zona Pellucida can be found. These conditions depend on the speed of cooling and warming, the type of storage container, and the cryoprotectant used. Careful observations of Zona Pellucida damage to embryos may therefore help experts to understand the reasons for its occurrence. It is therefore common practice to consider an embryo as surviving after Cryopreservation if at least half of the initial number of blastomeres remain intact. The respective importance of these most common criteria (blastomere survival, resumption of mitosis) that may help to select the best embryo to transfer after thawing, remains a matter of debate. It is therefore essential to use the right indications to allow the transfer of embryos with the best prognosis in order to enhance the possibility of pregnancy.

Outlining shortly semen samples Cryopreservation, it is important to note that spermatozoa have unusual cryobiological behaviour and improvements in their survival have not been achieved by the standard approaches of cryobiology. Conventional approaches to Cryopreservation impose a linear change of temperature with time; however, the stresses that cells encounter during Cryopreservation are all non–linear with time.

Improvements in Cryopreservation of spermatozoa have been attempted in the past by the use of different cryoprotectants and extenders, and in particular, by altering the cooling rate, usually a linear reduction in temperature with time [37; 42; 82]. Intriguingly, similar studies demonstrate that spermatozoa are relatively insensitive to the magnitude of the linear rate of cooling during freezing. With human spermatozoa, a very broad response curve exists with little difference in survival observed following cooling at 1°C/min up to 100°C/minute [42].

Although conventional models have suggested that sperm cells should survive cooling rates up to 10 000°C/min [77], experimentally the survival rate begins to decline beyond 100°C/min [42]. It is clear that spermatozoa have unusual cryobiological behaviour and improvements in their survival have not been amenable to conventional approaches of cryobiology. Therefore, the basic question for the Reproductive Andrology which should have been answered in the nearest future is the correlation between normalcy of the sperm, fertilization, and early embryo development and establishment of the detailed classification scoring scale for the individual spermatozoon with the highest predictive fertilizing potential in real time during Intracytoplasmic Sperm Injection (ICSI).

The additional purpose of this retrospective study was therefore to investigate the respective importance of oocyte damage, blastomere damage, spermatozoa damage and resumption of mitosis on the outcome of Cryopreservation.

1.  Oocyte [egg] Cryobanking [Embryo Cryopreservation]

What is Oocyte [egg] Cryobanking [Oocyte [egg] Cryopreservation]?

Oocyte Cryopreservation is a currently available method of Fertility Preservation or Cryopreservation of supernumerary oocytes in IVF/ICSI Treatment cycles.

Oocyte Cryopreservation had already gained more close attention as an option in infertility therapy [19; 40; 54; 81; 104]. During routine clinical Treatment, Ovarian Stimulation is carried out to increase oocyte production. Increasing success rates have led to more concerns regarding multiple gestation pregnancies. This has caused most clinicians to limit the number of embryos transferred during a cycle and to cryopreserve the supernumerary embryos and has revealed the problem of long–term storage of embryos and stimulated research trials on Oocyte Cryopreservation.

The development of efficient methods of Oocyte Cryopreservation would bring about a major breakthrough in In–Vitro Fertilization (IVF) Treatment Options. In fact, oocyte storage has the potential to circumvent several ethical and legal problems associated with Embryo Freezing, as well as to preserve female fertility in women at risk of premature ovarian failure, or in women who intend to postpone motherhood for various reasons.

More importantly, the possibility that the Oocyte Cryopreservation can become a valid option for preservation of fertility is inevitably dependent on the development of methods able to give success rates similar to Embryo Freezing. In this respect, despite the lack of controlled studies, it has been suggested that the efficiency of Oocyte Freezing is ∼50% compared with that guaranteed by cryopreserved embryos [14].

There are several reasons why oocyte Cryopreservation may be desired. 

Any condition which threatens to destroy all the follicles in both ovaries, or even destroy a large number of them, is an indication for Fertility Preservation. Both gynecological cancer and non–gynecological cancer may affect the ovarian reserve and ovarian function. The pathology itself may damage the ovarian tissues and its follicles. A possible application of Oocyte Cryopreservation techniques could be Fertility Preservation in women at risk of premature menopause, which can have several causes: recurrent or severe ovarian diseases such as cysts, benign tumors and endometriomas; ovary removal to treat endometriosis or genital cancer; and chemotherapy or radiotherapy to treat cancer or other systemic diseases. In addition, Oocyte Cryopreservation has been seen as a successful alternative for storing the excess oocytes during Assisted Reproduction Therapies, thus avoiding ethical, moral and religious dilemmas and reducing the number of embryos stored for future use. Couples undergoing Assisted Reproductive Treatment who do not wish to have embryos frozen for ethical or religious reasons could benefit from preserving excess oocytes for use in subsequent cycles.

The success of Oocyte Cryopreservation depends on morphological and biophysical factors that could influence oocyte survival after thawing. Various attempts to cryopreserve oocytes have been performed with contrasting results. Therefore, the effect of some factors, such as the presence or absence of the cumulus oophorus, the sucrose concentration in the freezing solution and the exposure time to cryoprotectants, on human oocyte survival after thawing were investigated. The oocytes were cryopreserved in 1,2–propanediol added with sucrose, using a slow-freezing–rapid–thawing programme.

Oocytes can be cryopreserved by two main methods: the slow–cooling computer–controlled Protocol and the ultrarapid cooling (Vitrification) Protocol. 

The results of Oocyte Cryopreservation using the Slow Freezing/Rapid Thawing (SF/RT) Protocol with 1,2–Propanediol (PROH) and high sucrose concentration (0.2 or 0.3 M) as cryoprotectants have shown a gradual improvement in efficiency over time, with live birth rates per transfer increasing during previous decade [48]. Vitrification seems to be a very promising technique even if, being a relatively new approach to Oocyte Freezing, a limited number of live births had been reported twelve years ago [79].

1.1. What phases does Oocyte Cryopreservation process include?

Collection of oocytes for diagnostic purposes [Pre–cryopreservation procedure]

The oocyte Pre–cryopreservation procedure consists of three phases: 

1. Controlled Ovarian Stimulation; 
2. Oocyte Retrieval and 
3. Oocyte Selection. 

1.1.1.  Controlled Ovarian Stimulation

Controlled Ovarian Stimulation [an efficient technique used in Assisted Reproduction involving the use of Fertility Medications to induce ovulation by multiple ovarian follicles] is usually achieved using GnRH analogues [GnRH –Gonadotropin–Releasing Hormone] in combination with a graded menotropin administration [31].

Dose adjustment of Fertility Medications is prescribed according to ovarian response. Currently, there was established a growing consensus to support a fixed daily injection Protocol starting on day 6 or 7 of the menstrual cycle (i.e. 5–6 days after initiation of stimulation) [1; 3; 34]. Final Oocyte Maturation should be induced by administering 6500 IU of hCG [hCG – human Chorionic Gonadotropin], when at least one follicle of ≥18 mm in diameter and two follicles of ≥16 mm in diameter were visualized by ultrasound. Follicular growth is monitored by serum oestradiol–17β measurements and ovarian ultrasonography.

1.1.2. Oocyte Retrieval

Follicle growth is assessed by transvaginal ultrasound, starting from CD 6 and thereafter as often as necessary in order to ensure that hCG [hCG – human Chorionic Gonadotropin] would be administered when the criteria has been met, with the possibility of postponing hCG [hCG – human Chorionic Gonadotropin] triggering by maximally 1 day. Transvaginal ultrasound guides Oocyte Retrieval is usually performed 35 hours after ovulation induction with 10 000 IU hCG [hCG – human Chorionic Gonadotropin] under general anaesthesia and results in the retrieval of 10–25 oocytes.

1.1.3.   Oocyte Selection [initial microscopic oocyte examination]

After Retrieval Oocytes are cultured for between 1 and 5 hours at 37°C in an atmosphere of 6% CO2 before the complete removal of cumulus mass and corona cells by enzymatic digestion with recombinant hyaluronidase and by gentle mechanic aspiration with plastic pipettes. The denuded oocytes are then evaluated to assess their nuclear maturation stage. The oocytes that have released the first Polar Body (metaphase II—MII) underwent a strict selection by morphological features (Zona Pellucida thickness, perivitelline space size, oocyte shape, cytoplasm colour and granularity, presence of vacuoles and first polar body morphology) under an inverted microscope with Hoffman modulation contrast. Those which are colorless and of regular shape, with regular Zona Pellucida and small perivitelline space without debris, homogeneous cytoplasm and no vacuoles or granulations are classified as ‘high quality’ oocytes [24; 28; 105]. Among the ‘high quality’ oocytes, the presence of an intact, round or ovoid Polar Body with smooth surface was considered as a selection criterion [27]. Only the supernumerary MII oocytes reaching the experts’ ‘high quality’ standards are cryopreserved.

1.2. The short description of the Oocyte Cryopreservation procedure

Usually, oocytes can be cryopreserved up to several hours after retrieval, depending on the laboratory procedures of each ART centre. After thawing, the oocytes should be cultured for a few hours before insemination to better evaluate the survival after the freezing/thawing procedure [39] and to allow the temperature–sensitive meiotic spindle to fully restore [13; 87]. The timing of ICSI is one of the important factors determining embryo viability and implantation: the developmental capacity of the oocyte declines 10 hours after Oocyte Retrieval [106]. The optimal timing for insemination of fresh oocytes seems to be 37–41 hours after hCG administration to trigger ovulation [25]. Similarly, the extent of metabolic ageing at ICSI of slow cooled oocytes depends on time of retrieval after hCG administration and on pre–incubation, but also on the post thawing culture before Insemination. Furthermore, it may be that the freezing procedure could influence cellular ageing.

The Oocyte Cryopreservation procedure consists of Cryopreservation Protocol and freezing procedure. 

Cryopreservation Protocol [exemplification of Cryopreservation Protocol]

The Cryopreservation Protocol consists of a SF/RT method. Oocyte Freezing and thawing solutions usually contain Dulbecco’s Phosphate Buffered Saline (PBS) supplemented with human serum albumin, alpha– and betaglobulins, and PROH and sucrose as cryoprotectants.

Freezing procedure [exemplification of freezing procedure]

After washing in a PBS solution, the oocytes are equilibrated for 10 minutes at room temperature in 1.5 M PROH and then transferred into the loading solution of 1.5 M PROH and 0.3 M sucrose. Between one and three oocytes are loaded in plastic straws and transferred into an automated biological vertical freezer. The cooling process is initiated reducing chamber temperature from 20°C to −7°C at a rate of 2°C/min. Ice nucleation is induced manually at −7°C. After a hold time of 10 minutes at −7°C, the straws are cooled slowly to −30°C at a rate of 0.3°C/minute and then rapidly to −150°C at a rate of 50°C/min. After 10–12 min at stabilization temperature, the straws are transferred into Liquid Nitrogen and stored for later use.

The metaphase II oocyte appears to be particularly susceptible to freeze–thaw damage, and it has been suggested that several forms of cryoinjury are responsible for the relative lack of success in preserving oocytes. These include damage to the meiotic spindle and to unstably bound chromosomes [38; 67; 89; 102;], to the microfilaments essential for Polar Body extrusion, pronuclear migration and cytokinesis [103], to the Zona Pellucida such as breaches and hardening [50; 97], and to the cortical granules causing a premature cortical reaction [2; 38; 90; 103].

1.3. Which factors can affect oocyte survival rates after Cryopreservation? [Scientific exemplification of factors]

Oocyte survival rates after Cryopreservation could be affected by morphological and biophysical factors. 

Morphological characteristics of oocytes such as maturity and size, and biophysical factors such as cryoprotectant compositions are particularly important. Among the morphological factors, the presence or the absence of the cumulus oophorus seems to play an important role in oocyte survival after thawing. It was postulated that the cumulus cells may offer protection against the adverse effect of the cryoprotectant and/or cooling in a way not yet explained [43]. In the literature, few investigators have focused their attention on the effect of denuding the oocytes of their cumulus cells before Cryostorage. However, the results reported in the few studies involving a low number of oocytes are controversial [43; 38; 69].

The main biophysical factor affecting the oocyte survival is the intracellular ice formation that generally pierces the membrane causing cell lysis. Because the oocyte is a large cell containing a large quantity of water, it requires a long time to reach adequate dehydration (osmotically balanced by the cryoprotectant solution) before lowering the temperature and thus it is more difficult to avoid ice crystal formation. Intracellular ice formation can be affected by the presence of the cryoprotectants in the freezing solutions, and by the freezing and thawing rate [92].

It is suggested that satisfactory Oocyte Dehydration should be obtained before lowering of the temperature; this could further avoid the formation of intracytoplasmic ice crystals which are the main factor influencing the oocyte survival rate during Cryopreservation procedures.

The cryoprotectants generally used in Oocyte Freezing Protocols are 1,2–propanediol (PROH, membrane–permeating cryoprotectant) and sucrose (membrane–non–permeating cryoprotectant). Their protective action is very complex and attributable to a number of properties [92], the most important of which is the beginning of the dehydration process. In particular, sucrose does not enter the cell, but exerts its beneficial effects by causing cellular dehydration through changes in osmotic pressure [23]: the increase of the extracellular solute concentration generates an osmotic gradient across the cell membrane, which draws water out of the cell, causing the cell to dehydrate before the freezing procedure. A 0.3 mol/l sucrose concentration causes a more adequate loss of intracellular water without excessive oocyte shrinkage which could lead to the collapse of the cellular membranes.

The presence of cryoprotectants (both permeating and non-permeating) in the freezing solution should minimize cell damage during the freezing and thawing process. For Oocyte Cryopreservation procedures, cryoprotectant concentrations are usually ~1.5 mol/l, many times higher than any other component in the medium. Thus, the cryoprotectants enter the cell by osmosis. While the cryoprotectants readily cross the cell membranes, water usually crosses even more readily [92].

Furthermore, it is extremely important to establish what the optimal exposure time of the oocyte to cryoprotectant solutions is. It has to be long enough to permit sufficient dehydration of the cell, but not so long as to damage the cell since it alters the intracellular pH [22]. It was suggested that an exposure time of 10 minutes could be suitable for improving the survival rate of oocytes [2].

Contrasting results are reported in the literature regarding the oocyte survival rate after Cryopreservation with or without the cumulus oophorus and using a solution consisting of 1.5 mol/l PROH to which 0.1 mol/l sucrose was added. For oocyte Cryopreservation with the slow freezing method, cryoprotectant solution usually consists of 1.5 mol/l membrane–permeating cryoprotectant (i.e. propanediol) and 0.1–0.3 mol/l sucrose. Post–thaw survival is no guarantee of unaltered developmental ability, and sperm microinjection may resolve fertilization failure originating from zona hardening, but poor fertilization may be caused by a myriad of other types of cell injury due to freezing. A significantly higher survival rate is obtained when the oocytes are cryopreserved in the presence of a doubled sucrose concentration (0.2 mol/l) in the freezing solution and the survival rate is even higher when the sucrose concentration was tripled (0.3 mol/l) (60 versus 82% P < 0.001). Furthermore, a longer exposure time (from 10.5 to 15 min) to cryoprotectants, before lowering the temperature, significantly increases the oocyte survival rate (P < 0.005).

It was observed [69] that the presence of the cumulus mass or the partial or the total removal of cumulus cells did not significantly modify the oocyte survival rate (36, 20 and 44% respectively). By contrast, it was found that the oocytes surrounded by a total cumulus and corona mass, as retrieved at ovum recovery, had a significantly reduced survival rate (48%) compared with those oocytes which had the mass removed prior to freezing (69%), suggesting that the presence of cumulus cells and the cumulus matrix causes a different rate and extent of dehydration during Cryopreservation [38]. The cumulus–corona complex may also form a more rigid structure limiting the distortion of oocyte shape which occurs during ice formation in the cytoplasm [8]. In conclusion, it is suggested that the presence of the cumulus oophorus does not affect the oocyte survival rate; the presence of a higher sucrose concentration in the freezing solution, and a longer exposure time to the cryoprotectant (under particular conditions) positively affect the oocyte survival rate.

2. Embryo Cryobanking [Embryo Cryopreservation]

What is Embryo Cryobanking [Sperm Cryopreservation]?

Embryo Cryopreservation plays a significant role in Assisted Reproduction Technologies (ART). In–Vitro Fertilization is associated with a high rate of multiple pregnancies, a consequence of the number of embryos transferred. During a typical In Vitro Fertilization (IVF) or Intracytoplasmic Sperm Injection (ICSI) treatment cycle, the woman’s ovaries are stimulated to produce many eggs. These eggs are then harvested, by placing a needle that goes into the follicle containing each egg. (This is done under general anesthesia). Originally, each oocyte is placed in its own dish, and the sperm put into the dish, to try to achieve fertilization. This is called conventional In Vitro Fertilization (IVF), and each oocyte needs about one million moving sperm to have a reasonable chance for fertilization to take place. Since multiple oocytes (eggs) are often produced during Ovarian Stimulation, on occasion there are more embryos available than are considered appropriate for transfer to the woman’s uterus. If viable, these embryos can be frozen for future use. Therefore, it is proven that elective Single Embryo Transfer closely correlates with the exceptional value of Cryopreservation. 

A new strategy is needed to improve the quality of Assisted Reproductive Techniques. The main reason for adverse Treatment outcome in Assisted Reproductive Techniques (ART) is the high rate of multiple pregnancies, including twins [12]. To avoid triplet pregnancies, two decades ago, many European IVF clinics have accepted the policy of transferring only two embryos [17], and nowadays an overall pregnancy rate of 30–40% per cycle is obtained in many Assisted Reproductive Technique programmes.

There is a challenge in avoiding even twin pregnancies in Assisted Reproduction, and this can be accomplished with elective Single Embryo Transfer and a perfect Cryopreservation programme, which is highly valuable.

The main indication for elective Single Embryo Transfer is the couple’s wish to avoid twins, and in some cases various medical reasons, which include diabetes mellitus, uterine malformation, a history of cervical incompetence and indication for prenatal diagnosis. If the multiple pregnancy is associated with high–risk for woman’s health, it is vital to emphasize, that one of the main challenges for fertility experts is to avoid twin pregnancies without significantly lowering the overall pregnancy rates. This can be done if the best embryo can be selected for transfer and if the freezing and thawing techniques can be improved. If strict criteria for selecting top quality embryos are utilized, higher ongoing pregnancy rates can be achieved.

Correct counselling is very important, as some infertile couples are known to desire multiple pregnancies. Good counselling should include realistic information, not only on the risks of twin gestation but also on later burdens with a multiple birth.

Cryopreservation of embryos produced during In Vitro Fertilization (IVF) or Intracytoplasmic Sperm Injection (ICSI) Treatment cycle is a logical way to provide an opportunity for a woman to have repeated attempts at conception following a single drug stimulation cycle, preventing wastage of valuable genetic material and improving cumulative pregnancy rates. Cryopreservation of embryos following IVF/Intracytoplasmic Sperm Injection (ICSI) provides further possibilities of pregnancy in addition to those achieved from the fresh cycle.

This approach may have several advantages: firstly, it provides an opportunity to limit the number of embryos transferred to the woman’s uterus while maximizing the usable embryo per Oocyte Retrieval cycle ratio at each stimulation attempt, a procedure that is potentially difficult for a woman. Secondly, the number of drug stimulation cycles in order to obtain oocytes can be decreased; consequently, the potential risk to the woman from exposure to anesthesia and the possible development of Hyperstimulation Syndrome can be reduced. In addition, storage of embryos from a cycle allows the woman to space the timing of sibling pregnancies, and improve their potential to achieve a pregnancy at an advanced maternal age, since the oocytes were retrieved when the patient was younger.

General information about Embryo Cryopreservation techniques

Two basic techniques have been employed for the Cryopreservation of cells: controlled slow–rate freezing and Vitrification.

Vitrification is a potential alternative to the conventional slow-freezing method used for Assisted Reproductive Technology (ART) – has high success rates; however, Vitrification/warming solutions usually contain high concentrations of HSA, which can cause biological variations and disease transmission.

Vitrification is becoming the method of choice for Embryo Cryopreservation. Nevertheless, major problems are still associated with this process such as chemical toxicity and osmotic stress as well as risk of Liquid Nitrogen (LN) contamination. An innovative Vitrification method that combines Liquid Nitrogen (LN) slush and Sealed Pulled Straws (SPS) was employed to achieve a high cooling rate, enabling a reduction in cryoprotectant concentration. Open pulled straws were sealed at both ends to prevent direct contact with Liquid Nitrogen (LN). This method enables maintenance of high cooling rates as well as reduction of cryoprotectant concentration, despite the use of a sealed container that helps to reduce the potential risk of contamination.

Vitrification is an ultra–rapid method of Cryopreservation whereby the embryo is transitioned from 37°C to −196°C in <1 s, resulting in extremely fast rates of cooling (>10 000°C/min). High concentrations of cryoprotectants together with rapid cooling rates are essential to cryopreserve embryos in a vitrified, glass–like state [99]. To facilitate rapid heat transfer, minimal volumes are used in Vitrification, facilitated through the use of minute tools as carriers. The carrier systems that have been developed for the Vitrification procedure include the electron microscope grid [80; 93], pulled and hemi–straws [29; 100; 101], flexipipet [58], cryotop and cryotip [55] and the cryoloop [56; 73; 74; 75; 85; 86]. The use of each technique has recently been reviewed.

The main advantages of Vitrification include the lack of ice crystal formation, made possible through increased speed of temperature conduction, reducing associated chilling injuries. Vitrification requires minimal set up time, being performed as needed during the course of the day. Furthermore, there is no need for expensive programmable freezing equipment. One potential disadvantage of vitrification is that it is considered technically more challenging than slow freezing and requires more hands–on time per cryopreserved embryo.

2.1. What phases does Embryo Cryopreservation process include?

2.1.1.  Controlled Ovarian Stimulation

Women are stimulated with either the long Gonadotrophin–Releasing Hormone (GnRH) agonist or a GnRH antagonist Protocol combined with recombinant follicle–stimulating hormone. Controlled Ovarian Stimulation [an efficient technique used in Assisted Reproduction involving the use of fertility medications to induce ovulation by multiple ovarian follicles] is usually achieved using GnRH analogues [GnRH –Gonadotropin–Releasing Hormone] in combination with a graded menotropin administration [31].

Dose adjustment of Fertility Medications is prescribed according to ovarian response. Currently, there was established a growing consensus to support a fixed daily injection Protocol starting on day 6 or 7 of the menstrual cycle (i.e. 5–6 days after initiation of stimulation) [1; 3; 34]. Final oocyte maturation should be induced by administering 6500 IU of hCG [hCG – human Chorionic Gonadotropin], when at least one follicle of ≥18 mm in diameter and two follicles of ≥16 mm in diameter were visualized by ultrasound. Follicular growth is monitored by serum oestradiol–17β measurements and ovarian ultrasonography.

2.1.2. Oocyte Retrieval

Follicle growth is assessed by transvaginal ultrasound, starting from CD 6 and thereafter as often as necessary in order to ensure that hCG [hCG – human Chorionic Honadotropin] would be administered when the criteria has been met, with the possibilityGJK of postponing hCG [hCG – human Chorionic Gonadotropin] triggering by maximally 1 day. Transvaginal ultrasound guides Oocyte Retrieval is usually performed 35 hours after Ovulation Induction with 10 000 IU hCG [hCG – human Chorionic Gonadotropin] under general anaesthesia and results in the retrieval of 10–25 oocytes.

2.1.3. Oocyte Selection [initial microscopic oocyte examination] 

After Retrieval Oocytes are cultured for between 1 and 5 hours at 37°C in an atmosphere of 6% CO2 before the complete removal of cumulus mass and corona cells by enzymatic digestion with recombinant hyaluronidase and by gentle mechanic aspiration with plastic pipettes. The denuded oocytes are then evaluated to assess their nuclear maturation stage. The oocytes that have released the first Polar Body (metaphase II—MII) underwent a strict selection by morphological features (Zona Pellucida thickness, perivitelline space size, oocyte shape, cytoplasm colour and granularity, presence of vacuoles and first polar body morphology) under an inverted microscope with Hoffman modulation contrast. Those which are colorless and of regular shape, with regular Zona Pellucida and small perivitelline space without debris, homogeneous cytoplasm and no vacuoles or granulations are classified as ‘high quality’ oocytes [24; 28; 105]. Among the ‘high quality’ oocytes, the presence of an intact, round or ovoid Polar Body with smooth surface was considered as a selection criterion [27].

2.1.4. Examination oocyte’s morphology of the Zona Pellucida surface by scanning electron microscopy

Oocytes from the same as well as from different women have an extremely heterogeneous morphology of the Zona Pellucida surface as shown by scanning electron microscopy. Analyzing the scientific theories, it was revealed that for years it has been believed that this heterogeneous morphology plays an important part in the sperm–oocyte interaction. It was the major aim of scientific investigations to analyze the morphology and the sperm binding patterns of the oocyte Zona Pellucida.

Usually, for this investigation, experts divide oocytes into four categories: mature, immature, fertilized and unfertilized. Four different types of zona morphology can be detectable through such kind of investigations. Types of zona morphology ranged from a porous, net–like structure to a nearly smooth and compact surface. It was scientifically proven that no correlation could be established between zona type and oocyte maturity or zona type and achieved fertilization. However, fertilized (polyploid) oocytes have a more compact and smooth zona surface than unfertilized ones. The analysis of the number and distribution patterns of bound spermatozoa on the Zona Pellucida revealed extremely variable patterns regardless of the zona morphology. Significant differences between mature and immature oocytes did not appear [66].

The Zona Pellucida is an extracellular matrix which surrounds the growing as well as the mature oocyte. It is composed of three different glycoproteins (ZPA, ZPB, ZPC) which together build a structure that varies greatly between different oocytes. This heterogeneity becomes particularly clear when examining scanning electron microscope photographs. Motta et al. (1991) [72] and later Harris et al. (1994) [41] described the zona of a mature oocyte as a network with multiple pores and hollows which is created by a three–dimensional arrangement of filaments. The porous structure might be the result of foot–like cytoplasmic branches from granulosa cells of the surrounding corona radiata, penetrating the Zona Pellucida to come in close contact to the plasma membrane of the oocyte during oogenesis. Apart from this network–like appearance a more compact and smooth surface has also been described. According to Sundström (1982) [95] this type can be found on non–ovulatory, immature oocytes.

The Zona Pellucida structure is classified in mature and immature as well as fertilized and unfertilized IVF and ICSI oocytes. An oocyte is classified as mature if, under the light microscope, one Polar Body was clearly visible in the perivitelline space, and immature if no Polar Body could be detected and/or the germinal vesicle is visible inside the cytoplasm.

Through the scientific investigation, the experts have distinguished four major types of the oocytes’ Zona Pellucida surfaces: 

1. Type, which represents a distinct net–like structure made out of numerous pores and hollows; 
2. Type, which represents a net–like structure which is composed of pores and hollows, flatter and of smaller diameter; 
3. Type, which has an uneven and spongy surface with very few or no porous areas and type, which is characterized by a relatively smooth exterior of the Zona Pellucida (pores and hollows hardly occurred and are scattered only in certain areas). 

When the surface of either oocyte’s Zona Pellucida type 1 or 2 was enlarged 10 000 to 20 000 times, an infinite number of small beads (80–130 nm in diameter), lined up like pearls on a string, became visible. In some parts of the zona these pearls formed clearly visible filaments which were also recognizable in the depth of the pores. In oocyte’s Zona Pellucida type 3 these pearl string–like filaments can be also clearly visible. However, their arrangement appeared less organized. If these little beads represent the basic construction material of the oocyte’s Zona Pellucida then it must be present in all zona types regardless of their appearance. So presumably in types 3 and 4 a conversion of the pearl string–like material must have taken place, especially in type D where the beads appeared to have melted into each other and were hardly recognizable (not shown). On certain oocytes still surrounded by some attached granulosa cells, cytoplasmic filaments from these cells penetrated the surface of the Zona Pellucida. When the oocytes’ with different Zona Pellucida morphology were compared [the number of mature and immature oocytes was differed] a significant correlation between the state of oocyte maturity and the surface structure of the Zona Pellucida was not found [66].

2.1.5.  Fertilization Process: sperm binding pattern

The goal of fertilization is the union of one, and only one, sperm nucleus with the female pronucleus within the activated oocyte. For this to occur successfully, several events must transpire, including the incorporation of the entire spermatozoon into the oocyte: during fertilization process spermatozoa have to recognize the Zona Pellucida and to bind to it (sperm–oocyte interaction). The initial binding of acrosome–intact spermatozoa occurs through terminal α– and β–galactose of the ZPC [60]; other zona proteins are also involved in this primary binding [66], the completion of meiotic maturation with the extrusion of the second Polar Body, the metabolic activation of the previously quiescent oocyte, the decondensation of the sperm nucleus and the maternal chromosomes into the male and female pronuclei respectively, and the cytoplasmic migrations of the pronuclei, which bring them into apposition. Defects in any of these events are lethal to the zygote and might prove to be causes of infertility.

Conventional In–Vitro Fertilization (IVF) is performed routinely 6 hours after Oocyte Collection (Day 0). Motile sperm concentration should be 0.5–1.0 × 106/ml according to sperm quality. Intracytoplasmic Sperm Injection (ICSI) was performed 4–5 hours after oocyte collection. Denudation of oocytes is performed by gentle pipetting after a short incubation in 80 IU/ml hyaluronidase. After sperm micro–injection, the oocytes are placed into culture media individually.

***Additional information, concerning investigation of sperm binding pattern as an integrative component of the fertilization process (additional investigation/examination of sperm binding pattern)

According to the gold standard, the oocytes should be inseminated after sperm preparation (swim–up technique) with nearly 100000 spermatozoa. For the thorough analysis of sperm binding patterns, a three–figure code can be used. This code is based on the results of our own investigations made by light microscopy [70] and gave information about:

1. The number of bound spermatozoa; 
2. The distribution of the binding sites on the surface of the Zona Pellucida;
3. And the appearance of cluster–like attachments of spermatozoa on parts of the Zona Pellucida [66].

The three–figure code consists of the following numbers. First number = number of spermatozoa bound onto the zona; 0 = none; 1 = 1–10; 2 = 11–50; 3 = 51–100; 4 = >100. Second number = distribution patterns of the bound spermatozoa; 0 = no spermatozoa on the zona; 1 = regular; 2 = irregular. Third number = existence of sperm clusters on the zona; 0 = none; 1 = no sperm clusters; 2 = sperm clusters present.

How unfertilized oocytes after performing In–Vitro Fertilization (IVF) can be distinguished?

Thorough microscopic examination of oocytes after performing In–Vitro Fertilization (IVF) distinguishes two oocyte categories: fertilized oocytes and unfertilized oocytes according to sperm binding pattern. Unfertilized oocytes do not have any spermatozoa attached to the Zona Pellucida or have only very few bound spermatozoa. Only a few unfertilized oocytes have either between 51 and 100 spermatozoa attached to the Zona Pellucida or an uncountable number. The distribution patterns of spermatozoa on the Zona Pellucida (even or uneven distribution with or without cluster formation) are not related to the number of bound spermatozoa. In some oocytes, distinct areas on the zona without any spermatozoa can be clearly visible. Such areas can be seen even on oocytes with uncountable numbers of attached spermatozoa. The binding of spermatozoa onto the Zona Pellucida occurs in an even or uneven distribution. In the latter case sperm clusters can sometimes be seen. A cluster signifies the binding of a high number of spermatozoa on a clearly limited area of the zona [66].

How mature and immature oocytes after performing In–Vitro Fertilization (IVF) can be distinguished?

To find out if the degree of maturity have some influence on the sperm binding patterns, mature and immature oocytes can be analyzed after performing In–Vitro Fertilization (IVF). The percentage of mature and immature oocytes without any bound spermatozoa can be found in mature and immature oocytes, while oocytes with an uncountable number of bound spermatozoa can be found only in mature oocytes. Sperm clusters are recognizable on mature as well as immature ones. There are no statistically significant differences in the number of bound spermatozoa between mature and immature oocytes [66].

Can different Zona Pellucida morphologies lead to different sperm binding patterns oocytes? 

To find out if different Zona Pellucida morphologies can lead to different sperm binding patterns oocytes can be analyzed according to their zona morphology. Exemplifying this issue, we would like to represent the scientific investigation, which was performed by Magerkurth C., Töpfer–Petersen E., Schwartz P., Michelmann H.W.:

Oocytes (n = 50) with a net–like structure (types 1 and 2) were compared to those of Zona Pellucida types 3 and 4 with a smooth and compact structure (n = 36). All oocytes had been inseminated with normospermic ejaculates.

The results have shown that in both groups 54% of oocytes had <10 attached spermatozoa. All other distribution patterns also occurred in nearly the same percentages without significant differences. With regard to the Zona Pellucida morphology it was not clear why certain oocytes had a high number of bound spermatozoa while others had almost none. Distinct areas on all types of Zona Pellucida surfaces were detectable where either no spermatozoa existed or sperm clusters appeared [66].

How spermatozoon interacts with Zona Pellucida: ultrastructure of sperm–Zona Pellucida interaction

It was revealed very heterogeneous courses of gamete interaction and penetration of spermatozoa into the Zona Pellucida through Scanning Electron Microscope examination. Different phases of sperm fusion became visible. They ranged from an extremely superficial, loose attachment to the commencement of penetration and finally to a total fusion of the sperm head with only the tail remaining visible [66].

In most cases a flat, tangential attachment of the sperm head to the surface of the Zona Pellucida appears, followed by an intrusion into the Zona Pellucida in exactly this position. However, vertical binding with a penetration by the tip of the head first also occurs. Especially in oocytes where large numbers of bound spermatozoa (with or without clusters) are detectable, the vertical binding and penetration is the most usual way [66].

In oocytes with a net–like, porous structure of the Zona Pellucida, sperm heads very often disappear deeply into the pores so that only the tails are visible from the outside. The filaments, resembling a string of pearls, surround the sperm head as soon as it penetrates the Zona Pellucida [66].

Comparison of the Zona Pellucida morphology of fertilized and unfertilized oocytes

After penetration of the spermatozoon into the Zona Pellucida, the so–called Zona Pellucida reaction occurs which leads to a change in the chemical and physical characteristics of the Zona Pellucida. In connection with these biochemical changes, modifications on the Zona Pellucida surface can be expected, which might be visible in Scanning Electron Microscope pictures. Having observed and analyzed numerous scientific articles, scientists Magerkurth C., Töpfer–Petersen E., Schwartz P., Michelmann H.W. found out contradictory results: Familiari et al. (1992) [30] could not find any changes correlated to fertilization whereas Nikas et al. (1994) [76] reported a high correlation in the zona morphology between fertilized and unfertilized oocytes. According to their findings, fertilized oocytes had a compact surface (types 3 and 4) in contrast to unfertilized ones with a porous structure (types 1 and 2). Thorough analysis of fertilized and unfertilized oocytes could not prove if these structural differences were actually related to fertilization or were the result of other factors [66].

Comparison of the surface Zona Pellucida morphology of oocytes after IVF or ICSI Treatment

To answer the question if the surface morphology of the Zona Pellucida might be influenced by different In–Vitro Fertilization techniques oocytes after IVF Treatment can be compared to those after ICSI Treatment. Whereas the cumulus complex of IVF oocytes is dissolved by the enzymatic reaction of spermatozoa, ICSI oocytes are treated with hyaluronidase immediately after follicular puncture to get the same effect [66]. Scientific investigation was provided to compare the surface Zona Pellucida morphology of oocytes after IVF or ICSI Treatment by scientists Magerkurth C., Töpfer–Petersen E., Schwartz P., Michelmann H.W. If the handling of oocytes had some influence on the zona surface then different morphologies of ICSI and IVF oocytes must be expected. However, comparison of oocytes from both groups showed no differences. This is certainly evidence that all zona types and their different peculiarities are not exogenous side effects of the Treatments related to IVF, ICSI or Scanning Electron Microscope [66].

How sperm binding patterns can be investigated? How fertilized and unfertilized oocytes after performing In–Vitro Fertilization (IVF) can be distinguished? 

After IVF Treatment the sperm binding patterns on unfertilized as well as fertilized oocytes can be analyzed. Through the analysis of light microscope pictures all oocytes (fertilized and unfertilized ones) have an extremely heterogeneous sperm binding pattern. These patterns do not correlate with oocyte maturity or the occurrence of fertilization [70].

This theory was scientifically proved through the analysis of Scanning Electron Microscope pictures from 216 unfertilized oocytes also confirmed these results: the number and the distribution patterns of bound spermatozoa on the Zona Pellucida was highly variable [66]. These findings supported the data of several other investigations [11] which assumed that factors such as maturity of the oocytes [68], morphology of the Zona Pellucida, or anomalies of the spermatozoa [61] were the reasons for this variation.

Unfertilized oocytes as usual do not have any bound spermatozoa on the Zona Pellucida [61; 70]. In oocytes with >10 bound spermatozoa only 50% have an even distribution pattern. On all of the other oocytes spermatozoa bound in extremely heterogeneous ways. Areas totally free of any spermatozoa are close to those which are overloaded, with spermatozoa sometimes forming cluster–like arrangements [66].

And on the contrary, there can be found significantly more spermatozoa bound to the Zona Pellucida of fertilized oocytes than compared to unfertilized ones [66; 68].

In this connection, it is of interest to mention that in spite of the high number of motile spermatozoa used for In–Vitro Fertilization only a relatively small number bound to the Zona Pellucida. Perhaps Sundström (1982) [95] was correct when he suspected a so–called selection function of the Zona Pellucida. This leads to the conclusion that this “selection function” would be disturbed in all of those oocytes which are overloaded with bound spermatozoa.

Are there any differences in the number of bound spermatozoa between mature and immature oocytes?

Several investigators did not find any differences in the number of bound spermatozoa between mature and immature oocytes [11; 61; 62 96]. Scientists Magerkurth C., Töpfer–Petersen E., Schwartz P., Michelmann H.W. also could not find any such differences. However, they have established that only in mature oocytes were >50 bound spermatozoa detected [66].

In contrast to these findings Oehninger et al. (1991) [78] and Franken et al. (1994) [32] mentioned, in connection with the hemizona assay, that on mature oocytes significantly more spermatozoa bound than on immature ones. They assumed that the meiotic maturity was correlated with increased potential of sperm binding.

2.1.6. Development of the zygote to blastocysts

The quality of pronuclei is examined 17–18 hours after fertilization. Zygotes are scored on an inverted microscope at a magnification of ×400. Pronuclear morphology (Z–score) of zygotes is performed as described by Scott et al. (2000) [91]. The integrity rate of pronuclei (i.e. their morphology) can be a criterion of cryostability, developmental potential of the zygote and/or effectiveness of concrete Protocol of Cryopreservation (conventional freezing as well as rapid freezing).

The integrity rate of pronuclei after thawing can be distributed into two types: high and low. If after 10 minutes of thawing it was possible to clearly observe: 

1. A border of pronuclear membrane and 
2. At least half of the chromatin in a condensed form (nucleoli), this integrity rate is denoted as high. 

The rest of the zygotes which don’t develop to blastocysts are denoted as dead.

2.2. The short description of the Embryo Cryopreservation procedure.

Embryo Freezing (Cryopreservation) is a method of preserving the viability of embryos be carefully cooling them to very low temperatures (–196^oC). This is carried out in the laboratory using specialized freezing equipment and the embryos can then be safely stored in Liquid Nitrogen for extended periods.

The failure or success of Embryo Cryopreservation is dependent upon how successful or unsuccessful the removal of water has been from the individual cells of the embryo. If water is left in the cells, it forms crystals when frozen. These crystals act like knives and disrupt the inside of the cells of the embryo or “cut” through the outer layer or “membrane” of the cells. If this cutting or disruption has occurred, the embryo will not survive. In order to avoid the formation of the water crystals, a “cryoprotectant” is added which replaces most of the water inside the embryo. Under the proper conditions, the cryoprotectant will not form crystals and the embryo can safely withstand the drastic reduction in temperature required for cryogenic storage.

Two basic techniques have been employed for the Cryopreservation of cells: controlled slow–rate freezing and Vitrification. Cryopreservation can be performed at different developmental stages, for instance, zygotes at the two pronuclei (2PN) stage and embryos at the 4–8–cell stage.

The short overview–exemplification of Cryopreservation of two–pronuclear zygotes

The Protocol of Cryopreservation by direct plunging into Liquid Nitrogen is used for pronuclear zygotes. For this Protocol, the terminology ‘Vitrification’ is often used. However, at the relatively slow speed of cooling (–600°C/min) an ice–free state of Vitrification medium is not maintained through cooling and warming. The glass–formation at cooling and the stability of the amorphous state of solution at warming have been previously reported [10]. It was established, that even 40% Dimethyl Sulphoxide (DMSO), which is a better glass former than Ethylene Glycol (EG), has a critical cooling rate, to avoid ice formation, of 500°C/min, and a calculated critical warming rate, to avoid ice formation (Devitrification), of over one billion degrees per minute [10]. In the described solution, the carrier medium and serum solutes provide additional stability against ice formation, but 30% penetrating cryoprotectants are not able to prevent ice forming during the cooling, and especially described warming, conditions.

Osmosis plays a central role throughout all negative effects of the Cryopreservation process of pronuclear embryos [44]. In contrast with pronuclear zygotes that are vitrified, warmed and directly rehydrated with intense osmotic processes, are fully destroyed. Taking into account the fact that the saturation by cryoprotectants is also accompanied by osmotic processes, aseptic technology should include a step–wise method of saturation by cryoprotectants [45].

Pre–cooling Treatment (exposure in cryoprotectants) is performed by step–wise exposure in 12% (v:v), 25, 50 and 100% of the rapid freezing solutions for 2, 1, 1 min and 30–50 s, before plunging into liquid nitrogen, respectively. Cooling and thawing of embryos are performed in Cut Standard Straws (CSS) [46]. Cut Standard Straws is produced from standard insemination 0.25 ml straws which are cut at an angle approximately 45°. Rapid freezing medium with one or two zygotes is dropped into this cut part of the straw. After exposure in rapid freezing medium, one or two zygotes with 0.75 µl of this medium is aspirated to the tip of pipettor and transferred to Cut Standard Straws. The Cut Standard Straws are first loaded into 0.5 ml straws, which are closed at both sides using an ultra–sound sealer and plunged into Liquid Nitrogen with a cooling speed of 600°C/min.

Cryopreservation process of pronuclear embryos

Vitrification is a process in which liquids solidify without crystallization [63]. Compared with the slow cooling procedures, Vitrification methods are very rapid. Three key factors influence the probability of successful Vitrification: cooling and warming rates, the composition of the cryoprotectant solution which is reflected in the viscosity of the sample and sample volume. Increasing the cooling/warming rate [99], raising the cryoprotectant content or decreasing the sample volume will each increase the probability of vitrification [5]. Vitrification is an ultra–rapid method of Cryopreservation whereby the embryo is transitioned from 37°C to −196°C in <1 s, resulting in extremely fast rates of cooling (>10 000°C/min). High concentrations of cryoprotectants together with rapid cooling rates are essential to cryopreserve embryos in a vitrified, glass–like state [99]. To facilitate rapid heat transfer, minimal volumes are used in Vitrification, facilitated through the use of minute tools as carriers. The carrier systems that have been developed for the Vitrification procedure include the electron microscope grid [80; 93], pulled and hemi–straws [29; 100, 101], flexipipet [58], cryotop and cryotip [55] and the cryoloop [56; 73; 74; 75; 85; 86]. The use of each technique has recently been reviewed.

Freezing Protocol [Cryopreservation techniques]

1,2 Propanediol (PrOH) is used as a permeating cryoprotectant. Sucrose, which is a large molecule that osmotically promotes dehydration during cooling and protects against cell lysis when thawing, is used as a nonpermeating cryoprotectant. A phosphate–buffered solution was also used, so that all steps can be performed outside the incubator and at ambient temperature. Freeze Kit–1 (reference no. 10012; Vitrolife) is used as recommended by the manufacturer throughout the freezing procedure [98].

Only high–quality embryos (grade 1 or grade 2) having at least five blastomeres on day 3 after oocyte pick up should be cryopreserved, since the cryosurvival rate is related to the initial quality of the embryo [52].

Embryos were rinsed for approximately 2 minutes in Cryophosphate buffered saline, a phosphate buffer solution with 25 mg/mL human serum albumin. They were then gently placed in Freezing Solution 1 for 10 minutes. The cells of the embryo shrink and then reequilibrate in this solution. The embryos were moved across and loaded into straws that aresterile, nontoxic, and of high quality by attaching the straw to a 1–mL syringe which is connected to the straw through 1–cm silastic tubing. The straws are then attached to a sterile plug to avoid leakage into the straw and to the Liquid Nitrogen (LN2) tank during storage. A maximum of three embryos can be placed into each straw.

Straws are then placed in the freezing chamber at ambient temperature, and the program was commenced. Planer KRYO 10 Series III is used for Cryopreservation.

The freezing program can be as follows:

Starting temperature: 18 –25°C.

Step 1: 2.0°C/min to 7.0°C.

Step 2: Hold at 7.0°C for 10 minutes. Seed after 2 minutes.

Straws were manually seeded at 7.0°C with LN2–cooled forceps close to the cotton plug.

Step 3: 0.3°C/min to 30.0°C.

Step 4: 30.0°C to below 80.0°C (at least 10°C/min).

Straws are then removed and plunged into the LN2 storage tank immediately [98].

The technique previously described by Larman et al. (2007a) [57] can be also used for Vitrification and warming of cleavage–stage embryos. All Vitrification and warming procedures are performed at 37°C. The embryos are held in 1 ml of the base Holding solution for 5–15 min. One to two embryos are placed into the Equilibration solution for 2 minutes. Once the 2 minutes had elapsed, the embryos are placed into the Vitrification solution for 30 s. The Vitrification solution has the same composition as the Holding solution except that it contains 16% (v/v) EG, 16% (v/v) PROH, 10 mg/ml Ficoll and 0.65 M sucrose (RapidVit™ Cleave, Vitrolife). The embryos are loaded onto the cryoloop (Hampton Research, Aliso Viejo, CA, USA), transferring as little medium as possible, typically around 50 nl. The cryoloop is then loaded into the cryovial hold on a cryocane, which is submerged in Liquid Nitrogen [9].

For warming, the cryoloop is removed from the cryovial and dipped into Warming Solution 1. Embryos fall off the cryoloop and are moved through 1 ml volumes of a serial sucrose dilution in G–MOPS supplemented with 12 mg/ml HSA (RapidWarm™ Cleave, Vitrolife): Warming Solution 1 (0.65 M sucrose) for 30 s; Warming Solution 2 (0.325 M sucrose) for 1 minute; Warming Solution 3 (0.125 M sucrose) for 2 minutes and Warming Solution 4 (0 M sucrose) for 5 minutes. For subsequent embryo development, embryos are then moved into G2.3 (Vitrolife) for 24 hours (Embryo Transfer) or 48 hours (blastocyst assessment) [9].

Additional scientific information: Innovative Vitrification method, which is used to achieve a high cooling rate

An Innovative Vitrification method that combines Liquid Nitrogen (LN) slush and Sealed Pulled Straws (SPS) was employed to achieve a high cooling rate, enabling a reduction in cryoprotectant concentration. Open pulled straws can be sealed at both ends to prevent direct contact with Liquid Nitrogen (LN). This method enables maintenance of high cooling rates as well as reduction of cryoprotectant concentration, despite the use of a sealed container that helps to reduce the potential risk of contamination [88].

In the past, Vitrification was achieved by simply plunging the sample into Liquid Nitrogen (LN) at −196°C [83]. During this process, heat transfer from the sample into the Liquid Nitrogen (LN) leads to the evaporation of Liquid Nitrogen (LN) around the sample, resulting in the formation of a nitrogen gas layer, which acts as an insulator. This insulation impedes the heat transfer [20] and makes it impossible to achieve the high and uniform cooling rate that is thought to be particularly important during the initial stages of cooling through the ‘dangerous zone’: this is the temperature zone (from 25° to 0°C) within which the embryo is most prone to damage from chilling injury.

Increasing the concentration of the cryoprotectant increases the viscosity of the Vitrification Solution (VS), thereby reducing ice crystal formation during the cooling process. However, cryoprotectants tend to be toxic when used at concentrations that are effective for successful Vitrification. The toxicity and the osmotic pressure created by the high cryoprotectant concentration are highly damaging to the cells, and it is therefore crucial to restrict cryoprotectants to low concentrations. It has been suggested that increasing the cooling rate by decreasing the sample volume allows a reduction in cryoprotectant concentration [5; 107], and a method for increasing the cooling rate by utilizing LN slush has been described previously.

The third factor influencing the probability of successful Vitrification is the volume of the sample. Minimizing the volume of the sample decreases the likelihood of ice crystal formation and thereby promotes vitrification [5; 7; 107].

Cooling rate and temperature measurements

Cooling rate is examined by plunging different cryo–carrier systems into Liquid Nitrogen (LN) and Liquid Nitrogen (LN) slush. Measurements can be recorded by inserting a 50 µm diameter T–type thermocouple into a 0.5 µl drop of 100% VS inside SPS, Super Open Pulled Straws (SOPS) and 0.25 ml straws. The thermocouple should be inserted into the SOPS in a very accurate and precise way after placing the drop (0.5 µl of 100% VS) inside the straws. The cryo–carriers can be plunged into Liquid Nitrogen (LN) at −196°C or into Liquid Nitrogen (LN) slush at −210°C. The thermocouple must be connected to a data logger that delivers the data collected every 0.1 s to a personal computer where it would be recorded. Temperature is measured in the same manner during the sealing process, and thermal images can be acquired with a radiometric infrared camera [88].

Successful Embryo Cryopreservation is an important component for Assisted Reproduction Technology (ART). The vitrification method employs Liquid Nitrogen (LN) slush and Sealed Pulled Straws (SPS), which combine to ensure a rapid cooling rate under aseptic conditions. The minimal drop volume together with the high cooling rate enable a reduction in cryoprotectant concentration without increasing the likelihood of crystallization throughout Vitrification [107] and warming, and minimized exposure to the ‘dangerous zone’. An optimal Vitrification Protocol can be adopted for each developmental stage resulting in successful Vitrification and the maintenance of embryo viability.

During the process of Cryopreservation, cells are exposed to subphysiological temperatures and are thus vulnerable to chilling injury because their lipids undergo phase transition [36]. The lipid composition of the membrane strongly influences its properties in general and its resistance to thermal stress in particular [6]. Chilling injury, which contributes to the extensive damage that occurs during the process of Cryopreservation, is temperature–dependent; it may occur because of slow changes in membrane properties and integrity [36]. The cooling rate is a key factor in determining chilling resistance since it controls the duration of sample exposure to the temperatures within the ‘dangerous zone’. Previous results of blastocyst Cryopreservation have been too inconsistent [59] for routine use in IVF laboratories, emphasizing the difficulty of cryopreserving blastocysts. This may be related to the high sensitivity of blastocysts to subphysiological temperatures during cooling [94].

Analyzing the method of Embryo Cryopreservation in the presence of low concentration of vitrification solution with sealed pulled straws in Liquid Nitrogen slush (an attempt was made to cryopreserve embryos at this stage using the combined Sealed Pulled Straws (SPS) and Liquid Nitrogen (LN) slush method), led by four scientists: Saar Yavin, Adaya Aroyo, Zvi Roth, Amir Arav, we can establish that the level of embryos’ chilling injury decreases by the improved Cryopreservation: “…plunging Sealed Pulled Straws (SPS) into Liquid Nitrogen (LN) slush increased the cooling rate, thereby reducing the period embryos were exposed to low temperatures during their transition through the ‘dangerous zone’. This appeared to decrease chilling injury, as evidenced by the improved Cryopreservation of blastocyst–stage embryos. In particular, a higher survival (re–expansion) rate and higher proportion of live blastomeres were achieved as compared with blastocysts preserved under the slow cooling rate [88].

An increase in cooling rate may permit a reduction in cryoprotectant concentration [84], which, since cryoprotective solutions are toxic to embryos, might be beneficial for embryo survival. Procedures that increase cooling rates to above 20 000°C/min may offer the advantage of achieving vitrification at lower cryoprotectant concentrations. Establishing a suitable cryoprotectant concentration for each stage of embryonic development requires a delicate balance. On the one hand, using high cryoprotectant concentrations to increase the probability of Vitrification and to avoid recrystallization during warming is not recommended. On the other, decreasing the cryoprotectant concentration in order to reduce the chances of toxicity and osmotic shock will result in ice crystal formation. A suitable balance must be achieved in each and every Cryopreservation procedure, since optimal Vitrification conditions are an important factor in successful embryo survival during warming [107]. Durability of embryos to toxicity and osmotic shock caused by high cryoprotectant concentrations is dependent on the time of exposure: the period during which embryos are exposed to these cryoprotectants may be minimized when the method is applied by a skilled technician. A reduction in cryoprotectant concentration may be beneficial to the embryos since it will reduce time dependency and decrease both toxic and osmotic effects. Nevertheless, the reduction in cryoprotectant concentration must be moderate because low cryoprotectant concentrations may also cause devitrification and recrystallization during warming [88].

The results revealed in the scientific study “Embryo Cryopreservation in the presence of low concentration of Vitrification solution with sealed pulled straws in Liquid Nitrogen slush” show high survival rates of embryos at several developmental stages, after enclosure in Sealed Pulled Straws (SPS) and Vitrification in Liquid Nitrogen (LN) slush. Use of the Vitrification Cryopreservation method enabled the scientists to increase the cooling rate, thereby minimizing embryo exposure to the ‘dangerous zone’ of temperatures that cause chilling injury. Plunging embryos enclosed in Sealed Pulled Straws (SPS) into Liquid Nitrogen (LN) slush permitted increased cooling rates and led to successful Cryopreservation of embryos at all stages in the presence of low concentrations of cryoprotectant. This may allow a reduction in cryoprotectant concentration and thus, in cryoprotectant toxicity level. This technique could be modified to suit different stages of embryonic development. However, more studies are needed to examine the interactions of different cryoprotectant concentrations based on Ethylene Glycol (EG) and ultrarapid Vitrification of embryos at different stages. The tolerance of early stage embryos to Ethylene Glycol (EG) must be examined in combination with a crystal–free–formation cryopreservation method [88]. In addition, the potential risk of contamination from other samples stored in the same Liquid Nitrogen (LN) containers has been postulated [88].

2.3. Which factors can affect embryo survival rates after Cryopreservation? [Scientific exemplification of factors]

The individual cohort of embryos is taken to slow freezing or Vitrification arms according to a previously prepared computer–generated randomization list. Usually, only good–quality embryos having five or more equal–sized and evenly shaped blastomeres, with <20% fragmentation are cryopreserved. Cryopreserved embryos are subsequently cultured up to the blastocyst stage after thawing/warming. Embryos are considered to have survived if >50% of the blastomeres are intact or if they have at least three viable cells present at thawing, and showing at least one blastomere divided by 18 hours of post–thaw culture. Metabolic analysis of embryos cryopreserved with Vitrification technique is subsequently performed.

Concentrations of cryoprotectants and the embryo’s stage–specific sensitivity to them.

To exemplify factors which can affect embryo survival rates after Cryopreservation, we would represent a paragraph from the scientific research “Embryo Cryopreservation in the presence of low concentration of Vitrification solution with sealed pulled straws in Liquid Nitrogen slush”, performed by Saar Yavin, Adaya Aroyo, Zvi Roth and Amir Arav: “…an examination of blastocyst development after Vitrification and warming at the 2–cell stage showed a dramatic increase in the probability of Vitrification with an increase in the Cryopreservation concentration to 75%. With lower cryoprotectant concentrations however, the drop containing the embryos will recrystallize during warming and ice crystals will damage the cells. Embryos at different developmental stages that were vitrified at a final cryoprotectant concentration of 87.5% Vitrification Solution (VS), resulted in significantly different blastocyst formation rates; the vitrified 2–cell–stage embryos were the most sensitive and exhibited a lower potential to develop to the blastocyst stage. This emphasizes the embryo’s stage–specific sensitivity to cryoprotectants. Studies comparing Cryopreservation at different embryonic stages with different cryoprotectants showed a positive correlation between embryonic stage and blastocyst formation when embryos were vitrified in different cryoprotectants [VS containing 15% EG (v/v) and 15% (v/v) Dimethyl Sulphoxide (DMSO)]. For embryos cryopreserved in 4.5 M dimethyl sulphoxide, those at the blastocyst stage were most affected and their developmental rate was reduced relative to the other embryonic stages. Compared with other cryoprotectants, EG permeates better and is less–toxic to embryos [4] than DMSO. High osmotic pressure and toxicity after exposure to higher cryoprotectant concentrations appeared to damage embryonic development to different degrees, depending on the embryonic stage. Since Devitrification and recrystallization may also impair the rate of embryo survival, the conditions of the warming process are very important. The quantity and size of the ice formed during warming depends on severa1 factors, in particular the concentration and composition of the cryoprotectant VS and the rate of warming [15; 64]. Optimal warming is considered to consist of two phases: the first requires slow warming from the storage temperature of −196°C up to the glass transition temperature (Tg) of under −140°C, and the second phase requires rapid warming up to physiological temperature [16; 51; 65]. The Devitrification of a vitreous cytoplasm during warming has been found to be associated with the death of embryos warmed slowly after conventional Cryopreservation. However, this injury can be prevented when the rate of warming is high enough to prevent cytoplasmic devitrification [84; 88].

3. Sperm Cryobanking [Sperm Cryopreservation]

Spermatozoa have unusual cryobiological behaviour and improvements in their survival have not been achieved by the standard approaches of cryobiology. Conventional approaches to Cryopreservation impose a linear change of temperature with time; however, the stresses that cells encounter during Cryopreservation are all non–linear with time. In scientific article “A novel approach to Sperm Cryopreservation”, written by Morris G.J., Acton E. and Avery S., it is shown that improved methods of Cryopreservation may be developed by specifically manipulating the manner in which cells experience physical changes instead of imposing a linear temperature reduction. Several Treatments were compared: control of solidification to achieve constant ice formation with time was more damaging than the standard linear reduction in temperature. However, Treatments which followed a chosen non–linear concentration profile, referred to as “controlled concentration” allowed recovery of almost all the cells which were motile before freezing. The biophysical basis of these different responses was examined using the cryostage of a scanning electron microscope and freeze substitution and it was found that, surprisingly, all samples of spermatozoa in the frozen state were neither osmotically dehydrated nor had any visible intracellular ice. Viability on thawing did not appear to correlate with conventional theories of cellular freezing injury, which suggests that for human spermatozoa other factors determine viability following freezing and thawing [71].

Control of the cooling rate is often primitive: samples are commonly suspended in the vapour above Liquid Nitrogen, resulting in significant differences in cooling rate between different samples. The resulting straw-to-straw variation and loss of viability may not be important where sperm counts are normal, but in the case of oligozoospermic or asthenozoospermic samples these losses may be highly significant [71]. With the development of Intracytoplasmic Sperm Injection and the availability of techniques for surgical Sperm Retrieval, there is an increased need to store low numbers of sperm and therefore to improve freezing techniques in order to maximize survival [18].

Improvements in Cryopreservation of human spermatozoa have been attempted in the past by the use of different cryoprotectants and extenders, and in particular, by altering the cooling rate, usually a linear reduction in temperature with time [37; 42; 82]. Intriguingly, similar studies demonstrate that spermatozoa are relatively insensitive to the magnitude of the linear rate of cooling during freezing. With human spermatozoa, a very broad response curve exists with little difference in survival observed following cooling at 1°C/min up to 100°C/minute [42].

Although conventional models have suggested that human sperm cells should survive cooling rates up to 10 000°C/min [77], experimentally the survival rate begins to decline beyond 100°C/min [42]. It is clear that spermatozoa have unusual cryobiological behaviour and improvements in their survival have not been amenable to conventional approaches of cryobiology.

Many of the changes in physical properties which occur in an aqueous cryoprotectant following ice nucleation are not linear with temperature. Parameters such as the ice fraction, concentration of ionic species, osmolality, pH, viscosity and gas solubility, all vary in a non-linear manner with temperature [33]. In addition, the biophysical characteristics of cells which determine the response to freezing, for example the cellular permeability to water, also change in a non–linear manner with temperature. Conventional approaches to Cryopreservation thus impose a linear change of temperature with time whilst the stresses that cells are encountering are all non–linear with time. It is therefore appropriate to examine whether improved methods of Cryopreservation may be developed by specifically manipulating the manner in which cells experience physical changes rather than imposing a linear temperature reduction. In order to implement the required control of external conditions a new cell freezer has been specifically developed to achieve the desired Protocols [71].

What is Sperm Cryobanking [Sperm Cryopreservation]?

Cryopreservation is the process of freezing tissue. Sperm Cryopreservation entails depositing, processing, freezing and storing sperm at a cryobank. [A sperm cryobank is a facility that collects, stores and freezes the sperm]. The frozen sperm may be stored for a short period of time or a long period of time. Once it is frozen, the sperm is referred to as “Cryopreserved sperm”. The purpose of cryopreserving semen (sperm banking) is to help ensure the possibility of conception in the future. Cryopreserved sperm can be used in Artificial Insemination, In Vitro Fertilization, and other Assisted Reproductive Procedures.

Short–term Sperm Cryobanking [Sperm Cryopreservation]

Short–term Sperm Cryobanking is the depositing, freezing and storage of sperm at a sperm bank for less than one year. Cryobanked sperm is then used in Artificial Insemination, In Vitro Fertilization (IVF) and other Fertility Treatment procedures. Short–term Semen Cryobanking is recommended to preserve semen for deferred inseminations when an intimate partner is temporarily absent. It is also recommended in cases of oligozoospermia (low sperm counts) where multiple semen collections and pooling may be desirable for use in a single insemination. Short–term storage is also performed prior to Assisted Reproductive Technologies (i.e., In Vitro Fertilization, gamete intrafallopian transfer, etc.) to secure a good quality semen specimen for the prospective procedure.

Long–term Sperm Cryobanking [Sperm Cryopreservation]

Long–term Sperm Cryobanking is the depositing, freezing and storage of sperm at a sperm bank for more than one year. Cryobanked sperm is then used in Artificial Insemination, In Vitro Fertilization (IVF) and other Fertility Treatment procedures. Most commonly, long–term Semen Cryobanking is recommended to preserve semen for deferred inseminations in case man will be undergoing a Treatment for cancer which may impair his sperm production or quality (chemotherapy, radiation, etc.), will be taking any ongoing medications which may impair sperm production or quality (sulfasalazine, methotrexate, etc.), will be undergoing any procedure which could affect testes, prostate, or your ability to ejaculate (e.g. prostate resection, retroperitoneal lymph node dissection, etc.); will be undergoing a vasectomy. It is also recommended in cases of oligozoospermia (low sperm counts) where multiple semen collections and pooling may be desirable for use in a single insemination or there is a medical condition which is beginning to affect your ability to ejaculate (e.g. diabetes, multiple sclerosis, etc.). Long–term storage is also performed prior to Assisted Reproductive Technologies (i.e., In Vitro Fertilization, gamete intrafallopian transfer, etc.) to secure a good quality semen specimen for the prospective procedure.

Other reasons to consider sperm banking include:

Trans male to female adolescents should consider banking. Many, if not most, will ultimately undergo hormone Treatment that will stop sperm production. This may be irreversible, and even if it isn’t, they will be very reluctant to go back onto medications that dramatically increase their testosterone levels, which would be necessary for the initiation of sperm production. Thus, at the very beginning of the transition, their sperm should be banked.

Men with Klinefelter’s Syndrome (who have an extra chromosome) may have either low numbers of sperm in their ejaculate or none. However, many of them may be producing low levels of sperm in their testicles. Over time, their production drops off. If sperm are ever found in the ejaculate, they should be immediately cryopreserved. Many male infertility (andrology) experts feel that these men should undergo Testicular Sperm Extraction with banking.

Testicular cancer most commonly presents during adolescence, though it is rare. Sometimes these patients at the time of presentation, and even prior to surgery, have no sperm in the ejaculate (are azoospermia.) This may be because whatever process made their affected testicle more prone to cancer is also affecting the other, in terms of sperm production. It could also be that the cancer in one testicle is inhibiting the production of the sperm in the other testicle. Sometimes, at the time of the removal of the testicle with cancer, some of the tissue may be removed and examined for sperm production. If sperm is found, it could then be frozen for later usage. This, of course, needs to be coordinated with a specialist prior to the surgery.

What phases does Sperm Cryopreservation process include?

1. Collection of semen for diagnostic purposes [Pre–cryopreservation procedure]

The sample should be collected in a private room near the laboratory, in order to limit the exposure of the semen to fluctuations in temperature and to control the time between collection and analysis. After that, the specimen container is placed on the bench or in an incubator (37 °C) while the semen liquefies. The specimen container should be kept at ambient temperature, between 20°C and 37°C, to avoid large changes in temperature that may affect the spermatozoa after they are ejaculated into it. It must be labelled with the man’s name and identification number, and the date and time of collection.

2. Initial macroscopic semen examination and initial microscopic semen examination

The Semen Analysis allows experts to identify problems to be addressed in order to maximize the quality of the man’s semen. This may reduce the need for more complicated interventions for the female partner. It will also allow experts to rule out significant medical problems that may contribute to poor analysis results. Each subsequent specimen is analyzed microscopically prior to freezing to assess concentration, motility, forward progression, semen quality, and total number of moving sperm.

Sperm concentration and motility are assessed according to the methods described by the WHO, (1992). Viability is assessed by staining with 5% eosin (WHO, 1992).

Membrane function is assessed using the HOS test [49]. Briefly, an aliquot of spermatozoa is diluted 10:1 with hypo–osmotic medium (7.35g sodium citrate and 13.51g fructose in 1000 ml, 150 mOsm/kg), and incubated at 37°C for 30 min. Spermatozoa are scored for the presence or absence of swelling in the tail region.

Acrosome function is assessed using a modified ARIC test [21]. Briefly, washed sperm are incubated in 10 μM calcium ionophore, A23187. The presence or absence of the acrosome is identified by staining with FITC linked to Pisum sativum lectin, and viable spermatozoa identified using bis-benzimide (Hoechst H33258). A score is derived by subtracting the number of viable, acrosome–reacted spermatozoa in the control sample from the percentage of viable, reacted spermatozoa in the ionophore–treated sample.

3. The short description of the Sperm Cryopreservation procedure

Immediately after the specimen is analyzed, and prior to freezing, a special fluid (a cryoprotectant –– before the Cryopreservation procedures, semen samples are diluted with equal volumes of cryoprotectant) is added to aid the freezing process. This helps the sperm survive the freezing and the subsequent thawing process, which is performed when they are ready to be used or tested. The combination fluid (semen plus cryoprotectant) is then divided into portions and placed in separate vials. Each vial holds up to 2cc’s. A small amount of the combination fluid (specimen and cryoprotectant) is placed in a separate vial (test or T–vial). This is usually about 0.2 cc’s or less. The test tubes are gradually frozen. After 30–60 minutes they are transferred into Liquid Nitrogen tanks for permanent frozen storage. Frozen sperm must be stored in extremely cold temperatures –196°C (–321°F). The Liquid Nitrogen is independent of any source of power. Cryogenic tanks are checked daily and replenished as needed.

Samples can be frozen in 0.25 ml straws, sealed with polyvinylalcohol powder. For example, ten straws can be frozen using each experimental Protocol. Freezing can be carried out using a variety of methods [71]:

Method 1. Suspension in nitrogen vapour, 20–25 cm above the surface of Liquid Nitrogen in an open Dewar, no manual nucleation of ice.

Method 2. Within a vapour phase controlled rate freezer. Straws are held vertically and cooled from 20°C to –5°C at a rate of 2°C/min. Straws are maintained at –5°C for 10 minutes, during which time they are nucleated manually by touching the wall of each straw with forceps previously cooled in Liquid Nitrogen. Straws are then cooled at a programmed linear rate of cooling of 10°C/min to –100°C and then transferred to Liquid Nitrogen for storage.

Method 3. Using the Planer controlled rate freezer programmed as in Method 2, but with no manual nucleation of ice.

Method 4. In the Asymptote SF100 freezer (Cook IVF, Letchworth, Herts, UK), in which straws are held horizontally, programmed as in Method 2, with manual nucleation.

Method 5. In the Asymptote SF100 freezer as Method 4, but without manual ice nucleation.

Method 6. In the Asymptote SF100 freezer to control various extracellular physical conditions following nucleation of ice at –5°C .

After a minimum of 24 hours has elapsed from the time of the initial freezing, the test vial is thawed and tested again to ascertain from each specimen how well the sperm survived the freezing, in terms of number of sperm, percentage moving, and quality of the movement. Electron microscopy also can be used for thorough examination. Cross–fracture of the straws followed by deep etching to remove ice revealed the structure of the freeze–concentrated glycerol which has a uniform appearance across the straw. The revealed ice crystal structure shows a similar structure and spacing of ice crystals in all materials frozen by the different methods in which ice was manually nucleated at –5°C [71].

Usually, at higher magnification the freeze–etched samples can reveal that the sperm cells have migrated into the freeze–concentrated material during solidification: spermatozoa were not entrapped within ice crystals. However, some sperm tails are observed to extend away from the freeze–concentrated material, suggesting that these structures are associated with or entrapped in ice. Occasionally, spermatozoa are observed with the sperm head entrapped in one portion of freeze–concentrated matrix with the sperm tail tethered in a distinct zone with the intervening tail bridging a void that would have contained an ice crystal. Some spermatozoa are associated with the interface between the freeze–concentrated material and ice crystals, but generally few spermatozoa are apparent, but these spermatozoa do not appear to be osmotically shrunken. Occasionally, very distorted spermatozoa are observed; these are apparently entrapped between ice crystals rather than osmotically dehydrated [71].

Light microscopy of freeze-substituted sections can show that spermatozoa are entrapped within the freeze–concentrated material. Occasional spermatozoa are observed to bridge across two freeze–concentrated zones. At this magnification the freeze–concentrated matrix is observed to be relatively homogeneous in appearance following both linear cooling and linear ice fraction solidification. However, following “controlled concentration” areas of granularity are evident, which may be interpreted as substituted ice crystals within the freeze–concentrated matrix [71].

It was scientifically proved that all samples are observed to contain substituted ice crystals in the freeze–concentrated matrix. Frozen spermatozoa in the freeze-concentrated matrix are surprisingly similar to unfrozen controls. There is no evidence of osmotic shrinkage or of the presence of ice voids within the heads of the spermatozoa for any of the freezing methods examined. Occasionally, some shrinkage is observed in distal sections. Many sections of sperm head and tails are surrounded by a zone, less than 0.1 μm wide, of low electron density material [71].

Freeze fracture electron microscopy and freeze substitution extends previous observations made by light cryomicroscopy [53] that spermatozoa and solutes migrate either entirely into the freeze–concentrated matrix or are entrapped near to the interface of the ice and the freeze–concentrated material. In some cases sperm tails may be associated with ice crystals whilst the sperm heads are within the freeze–concentrated matrix. In extreme cases the head of the sperm is situated in one region of the freeze–concentrated matrix with the end of the sperm tail in another zone with the tail bridging through an ice crystal. This may occur because the dimensions of the freeze–concentrated matrix make it difficult to accommodate the sperm head and tail except when lying in the plane of the matrix, or because the tail section of the spermatozoon has surface properties which made it less likely to be excluded from the ice crystal matrix. It is important to note that the relative sperm cell recovery on thawing is not correlated with the structure of the ice crystal network because this structure is essentially fixed by the temperature of ice nucleation in the undercooled straws. All samples nucleated at the same temperature (–5°C) formed a similar initial ice structure upon which all additional ice was subsequently deposited [71].

Major differences in the eutectic structure between different treatments are made apparent by freeze substitution. Following “controlled concentration” freezing the freeze-concentrated matrix contained large ice crystals, which are absent from the matrix of the other less successful Freezing Treatments. Further studies are required to determine at what temperature these ice crystals form within the freeze-concentrated matrix, but it is apparent that sperm cells within this matrix are in close association with ice crystals. It is of interest that gross ice formation within the eutectic is observed in the sample with the highest recovery on thawing [71]. The establishment of spatial gradients within freeze-concentrated materials has been clearly demonstrated by light cryomicroscopy [53].

It has been demonstrated [26] that spermatozoa behave as ideal osmometers, within the range 250 to 1500 mOsm of sodium chloride and that 13% of the isotonic water is osmotically inactive. Using models of the osmotic behaviour of spermatozoa during freezing it has been suggested that following a linear cooling at 10°C/min, less than 10% of the cellular water would remain in the cell at –10°C. However, this major loss is not observed here: spermatozoa can exhibit no osmotic shrinkage. Although the low water content of sperm cells, combined with their flat, non–spherical shape, could allow large changes in cellular water content to cause little modification in the surface area, there is no evidence of any membrane alterations consistent with osmotic dehydration in any of the micrographs examined. However, if spermatozoa is frozen in the presence of glycerol, which has a high permeability to spermatozoa [35], it will reduce the water permeability of spermatozoa [77]. It is also of potential significance that an aquaporin (AQP7) which mediates water permeability in spermatozoa is also involved in glycerol transport [47]. The lack of cellular shrinkage would be consistent with the cells effectively being in equilibrium with glycerol at all temperatures during freezing. Similarly the apparent absence of intracellular ice  could also be associated with a high intracellular glycerol concentration.

Whilst experimental treatments give significantly different levels of viability any correlation with cell morphology in the frozen state is lacking, and it is the controlled parameter, namely the rate of change in solute concentration, which is the major factor affecting sperm recovery [71].

What is vital to know that although in the successful “controlled concentration [controlled rate of change in extracellular concentration]” method 6 of Sperm Cryopreservation the undercooling rises to a higher level initially, it remains approximately constant throughout the rest of the freezing whereas the linear cooling rate and linear ice cases rise significantly towards the end of the freezing process. This would be expected to increase the likelihood of intracellular ice. If instead the mass transport is dominated by the transport of glycerol that all cases remain close to equilibrium at the start, but the local undercooling subsequently increases. This increase is almost linear in the “controlled concentration” case but stays low and rises rapidly in the later stages [71].

However, it must be noted that intracellular ice is not apparent and it is therefore likely that other physical events determine viability during freezing and thawing. The corresponding estimated changes in mass transport for water movement and glycerol movement respectively. These values are again comparable for similar membrane permeabilities, but would scale relatively for different relative permeabilities. These estimates show the relative form of mass transport where the transport is dominated by either the water or glycerol transfer, and it would be expected that the cells may experience a combination of the two effects over the course of the freezing [71].

Cell viability may be determined by a combination of potential cytotoxic events at high sub-zero temperatures together with restrictions on transport at low temperatures due to low permeability, high viscosity etc. The “controlled concentration” treatment would minimize the time of exposure at high sub–zero temperatures and allow extended periods at lower temperatures to compensate for reduction in transport processes.

From these results, experts will be able to project with reasonable accuracy the quantity and quality of moving sperm that will be found in each of the storage vials when they are thawed for use in the future.

Conclusion

In conclusion, the present article demonstrated significant improvement in understanding the essence of Cryopreservation Technologies, especially, the methods of evaluating oocyte/embryo/sperm morphology based on very strict criteria, which allows a more accurate prediction of the chance of fertilization in vitro. Additionally, it is vital to emphasize, that technically, the possibility of offering highly efficient oocyte/embryo and Sperm Cryopreservation [Vitrification] needs not only modifications for system Vitrification and usage of high security closed vitrification devices, but also inclusive scientific investigation of the most delicate methods of whole Cryopreservation procedure and very accurate choice of the Cryopreservation technologies, considering individual morphological features and biophysical parameters [in every single case]. These data confirm that an efficient Cryopreservation program permits to give a substantial contribution to the cumulative pregnancy rate. Further studies should be done to establish the most appropriate cut–off points for severely impaired, intermediate and high fertilization rates.

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