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Doi:10.1016/j.rigp.2004.10.00

Reviews in Gynaecological Practice xxx (2004) xxx–xxx Cryopreservation of two pronuclear stage zygotes Yasser OriefNikos Safaa Department of Obstetrics and Gynecology, Shatby University hospital, Alexandria University, Egypt Laboratory of Reproductive Physiology, Faculty of Medicine, Demokritus University of Thrace, Dragana, 68100 Alexandroupolis, Greece Department of Obstetrics and Gynecology, Medical University of Lubick 23538, Germany Received 18 June 2004; accepted 11 October 2004 The German embryo protection law (Embryonenschutzgesetz, ESchG) does not allow embryo selection, but only selection at the pronuclear stage. Furthermore, only as many pronuclear stage zygotes are allowed to be selected as are planned to be transferred in the same cycle. This means that after pre-selection of, for example, three pronucleated zygotes, these three must be transferred on the same or the subsequent day. A second selection process is not allowed. Non-selected pronuclear stage zygotes are allowed to be cryopreserved for a The same situation is present in other European countries such as Swizerland and Italy. it is illegal to cryopreserve an oocyte after fusion of the pronuclei (PN). The idea of these laws was to avoid ethical problems related to cryopreservation of surplus embryos or wastage of embryos, because these have, according to these laws, the status of individual persons.
The current situation initiates much interest in developing a refined method of cryopreserving human pronuclear zygotes. The following article will discuss that issue in details.
# 2004 Published by Elsevier B.V.
Keywords: Cryopreservation; Two pronuclear stage zygote.
Cryopreservation of human embryos has been introduced into clinical IVF in order to preserve supernumerary A well-established embryo transfer (FET) embryos for a later transfer. Human embryos at different programme is essential in every assisted reproductive developmental stages have been frozen with variable success technology (ART) unit. Cryopreservation programs may rates. The pronuclear stage appears to be the optimal stage also increase the cumulative pregnancy rates of IVF and for cryopreservation The unicellular form and lack of spindle apparatus may account for its high post-thaw Freezing and storing of surplus embryos also allows the survival and implantation potential. Using this stage for number of replaced embryos in both fresh and frozen freezing, there are no ambiguities about whether embryos embryo transfers to be reduced, thereby diminishing the risk survive thawing because subsequent embryo cleavage of multiple pregnancies In addition, if the woman has a essentially proves cellular integrity. In addition, in some risk of developing ovarian hyperstimulation syndrome all European countries, the freezing of cleaved stage embryos is embryos can be cryopreserved . However, careful illegal as in Germany, Switzerland and Italy, thus limiting consideration of all clinical and embryological factors the choice to freezing of either unfertilized oocytes or influencing the outcome of FET is a prerequisite for a pronuclear stage zygotes.
Several protocols of freezing have been formulated depending on the embryo cellular stage, type of cryopro- tectant and speed of cooling.
Conventional (slow) freezing of human pronuclear * Corresponding author. Tel.: +49 451 500 2155; fax: +49 451 500 4904.
E-mail address: [email protected] (S. Al-Hassani).
zygotes has been the most widely used method of storage 1471-7697/$ – see front matter # 2004 Published by Elsevier B.V.
doi:10.1016/j.rigp.2004.10.001 UNCORRECTED PROOF

Y. Orief et al. / Reviews in Gynaecological Practice xxx (2004) xxx–xxx up until now with variable results Slow cooling constituted 2/5 of the maximum score. Scoring was done at procedures have the disadvantage in that they are time 16–18 h post ICSI according to (i) the position of the PN, (ii) consuming and require accurately controlled expensive the alignment of nucleoli at the junction of the two freezing units, making them unsuitable for use where cost pronuclei, and (iii) the appearance of the cytoplasm.
and time is a consideration. Different freezing protocols that In Germany, however, this item cannot be included, since are faster and cheaper and achieve higher survival and only selection at the PN stage is allowed and supernumerary development rates after freezing and thawing than do PN zygotes must be cryopreserved at the PN stage or conventional freezing procedures have been reported.
The rapid procedure for freezing human pronuclear embryos such as that reported by Trounson et al. has 1.1.2. Freezing and thawing procedures been reported by a few IVF groups with variable results The supernumerary Zygotes of the collecting cycles are cryopreserved 18 h after the IVF or ICSI procedure. Ham's However, there have been several recent reports of the F-10 (Biochrom Company, Berlin, Germany) supplemented successful cryopreservation of human pronuclear zygotes by with 20% human umbilical cord serum is used as freezing direct plunging into liquid nitrogen (vitrification) solution. The cryoprotectants 1,2-propanediol (PROH) and This method is now an object of intensive investigation in a sucrose are used at concentrations of 1.5 and 0.1 mol/L, number of laboratories, taking into account that the protocol respectively Pronuclear stage embryos are then of vitrification includes two major benefits: the complete equilibrated in two steps (first step: 1.5 mol/L PROH, process can be completed in only minutes in contrast to a second step: 1.5 mol/L PROH and 0.1 mol/L sucrose) at long time for the conventional method, and this method does room temperature, each for 10 min. A CTE-880 biological not require specialist equipment, in contrast to conventional freezer (Cryo Technik Company, Erlangen, Germany) slow freezing techniques.
working with an open freezing system and self-seeding Prior to the successful vitrification of human pronuclear was used for cryopreservation. Up to three 2PN zygotes are zygotes, vitrification of fertilized animal oocytes was transferred with medium to each ministraw (Cryo Technik developed by an effective protocol for the vitrification of Company). The ministraws are cooled slowly from room mouse oocytes, which involved direct plunging into liquid 33 8C. They should be kept at nitrogen Since then, several publications on the 30 min and then they are plunged directly into liquid vitrification of animal oocytes at the pronuclear stage have nitrogen for storage .
emerged, in which the ability of cells of transgenic mice The thawing procedure begins with the direct transfer of and rabbits to develop after cryopreservation was ministraws to a 30 8C water bath, for 30 s. After this, the evaluated. Subsequent protocols for the vitrification of cryoprotectants are diluted in four steps, using different human pronuclear zygotes were based on the data provided solutions: first, with 1 mol/L PROH and 0.2 mol/L sucrose; by these studies.
second, with 0.5 mol/L PROH and 0.2 mol/L sucrose; third, with 0.2 mol/L sucrose; and finally with Ham's F-10 1.1. Slow freezing technique medium alone. Each step should last 5 min Pronuclear stage zygotes are then cultured in Ham's F-10 1.1.1. Preparation of oocytes for 2–3 h and then inspected for survival under both a Following oocyte retrieval, the cumulus and corona stereomicroscope (magnification 50) and an inverted radiata are removed mechanically under a stereomicroscope, microscope (magnification 200–400).
after exposure to 0.5% hyalouronidase solution (Sigma Company, Deisenhofen, Germany) for 30 s. IVF or ICSI are 1.2. Ultra-rapid freezing technique performed as previously described by Al-Hasani et al. Pronucleate zygotes must have an intact zona pellucida The zygotes are again first exposed to a cryoprotectant, and healthy cytoplasm with two distinct pronuclei clearly equilibration prior to freezing is carried out as described in visible. When pronuclei start to migrate before syngamy the the slow freezing method. Zygotes are then drawn up into mitochondreal system is highly vulnerable to temperature plastic straws, also electron microgrids can be used as a fluctuation leading to possible scattering of the chromo- physical support, before they are plunged directly into liquid somes. Ludwig et al. recently published a new scoring nitrogen after 2–4 min.
system for zygotes at the PN stage. This score is based on the For thawing, the straw is gently expulsed into a phosphate fact that a faster developmental process after fertilization buffered solution containing 20% fetal calf serum and demonstrates a better quality of the zygotes and resulting 0.25 mol/L sucrose for 10 min at room temperature. The embryos. Their score included not only the morphological zygotes are then placed in culture and incubated for 2 to 4 h appearance of the pronuclei, but also the further develop- before transfer into the recipient uterus.
ment up to the PN membrane breakdown and first cleavage As freezing solution EFS30 is often used consisting of division. This last item (PN membrane breakdown and first 30% Ethylene Glycol, 18% Ficoll, 0.5 mol/L sucrose, 10% cleavage division within 24–26 h UNCORRECTED PROOF post oocyte retrieval) fetal bovine serum with added modified Dullbecco's

Y. Orief et al. / Reviews in Gynaecological Practice xxx (2004) xxx–xxx phosphate buffered saline, supplemented with sodium reaching the homogenous nucleation temperature. On the pyruvate (0.33 mmol/L), glucose (5.6 mmol/L), penicillin other hand, the slow freezing method requires expensive G (0.0375 g/L) and streptomycin(0.025 g/L).
equipment and is time consuming.
With the ultra-rapid freezing method the need for a 1.3. Vitrification computer controlled freezing apparatus is avoided and the time required for freezing and thawing is greatly reduced.
The physical definition of vitrification is the solidification However, the extreme toxicity of the high concentration of of a solution (water is rapidly cooled and formed into a the cryoprotectant solution is the main disadvantage of this glassy, vitrified state from the liquid phase) at low method. Van den Abbel et al. compared a slow temperature, not by ice crystallization but by extreme controlled rate freezing procedure with a rapid cooling elevation in viscosity during cooling This method procedure using one-cell human embryo. They showed that combines the use of concentrated solutions with rapid slow controlled rate freezing is more efficient than rapid cooling in order to avoid ice formation. The samples reach low temperature in a glassy state which has the molecular Vitrification can be an alternative to the conventional structure of a viscous liquid and is not crystalline.
slow freezing protocol with advantages of the lack of the ice Today, human pronuclear zygotes can be cryopreserved crystal formation and ease of operation. The method also has successfully by vitrification The efficacy of a the advantage of taling only a few seconds to cool embryos.
rapid freezing method using the electron microscope copper Furthermore, it does not require a controlled rate cooling grid or the Flexipet denuding pipette (FDP) for human PN apparatus. However, Uechi et al. by comparing the embryos has already been reported conventional slow controlled rate freezing and vitrifcation With respect to survival, cleavage on Day 2, and on two-cell mouse embryos, showed that the implantation blastocyst formation, a high survival and cleavage rate of rate of blastocysts developed in vitro from vitrified two-cell multi-pronuclear zygotes was also documented. Liebermann embryos was significantly lower than that from slow and Tucker using 5.5 M EG, 1.0 M sucrose, and an controlled rate frozen embryos (10.2% versus 22.1%).
FDP as a carrier for the vitrification, observed 90% of 2PN Vitrification may, therefore, exert a more harmful effect than survival after warming and 82% of 2PN cleavage on Day 2.
the slow controlled rate freezing in two-cell embryos. The On Day 3 in the vitrified 2PN group, approximately 80% of same could be also speculated for one-cell embryos.
embryos cleaved to become an embryo with four or more To date, vitrification as a cryopreservation method has blastomeres, and 30% of 2PN embryos eventually became had very little practical impact on human-assisted reproduc- tion. This may be due to the wide variety of different carriers More recently, successful pregnancies after vitrification and vessels that have been used for vitrification. Second, of human zygotes have been reported . It is stated many different vitrification solutions have been formulated, that the pronuclear stage is well able to withstand the which has not helped to focus efforts on perfecting a single vitrification and warming conditions. Probably, this might approach. On the other hand, the reports of successfully be due to the processes during and after the fertilization, completed pregnancies following vitrification at all pre- such as the cortical reaction and subsequent zona hardening implantation stages is encouraging for further research and that may give the ooplasmic membrane more stability to cope with the low temperature and osmotic changes.
Finally, the low toxicity of EG, together with the good survival, cleavage, blastocyst formation, and pregnancy 3. Assessment of embryo survival rates obtained after vitrification of pronuclear zygotes, may satisfy the real need in countries where cryopreservation of Since the only criteria to evaluate whether the zygotes later-stage human embryos is not allowed by law or for survived the freezing /thawing procedure is if they retain ethical reasons.
their pre-freeze morphology (e.g. PNs existence, have no obvious damage to the zona pellucida and oolemma, if their cytoplasm is clear and re-expands to its original 2. Comparison of the different cryopreservation volume after rehydration etc.), thus only if they cleaved in culture after 16–24 h, they are appropriate for intrauterine Because of the low water permeability and a low surface to volume ratio of the two pronucleate zygotes, a slow cooling rate may be advantageous. At slow cooling rates the 4. Preparation of transfer cycles compositional changes in the intracellular solution can follow those in the extracellular solution. Intracellular The success of frozen embryo transfer requires synchro- freezing is avoided because the water content of the zygote nization of the endometrium to enable it to receive embryos has approached the equilibrium UNCORRECTED PROOF water content before which have arisen from a different menstrual cycle. Transfer

Y. Orief et al. / Reviews in Gynaecological Practice xxx (2004) xxx–xxx of frozen thawed embryos may take place in a natural cycle concerns raise additional interest in the cryopreservation or alternatively in a programmed cycle with comparable outcome of embryos or oocytes.
pregnancy rates of 15–20%, respectively .
Nikolettos et al. in a retrospective study, compared In a natural cycle the patients are monitored for the onset the cryopreservation outcome of 2PN zygotes obtained by of endogenous luteinizing hormone (LH) surge (day 14) and cetrorelix and triptorelin depot. They reported 3.26% the transfer of the embryo is performed on Day 17.
implantation rate for the cetrorelix group and 3.73% for Alternatively, in programmed cycles, the endometrium is the triptorelin group, as well as pregnancy rates of 8.33 and exogenously stimulated with sequential estrogen and 10.25%, respectively. They concluded that there was no progesterone following down-regulation of the hypophysis negative effect of cetrorelix on viability, implantation with a gonadotrophin releasing hormone agonist (GnRHa) to potential or pregnancy outcome.
prevent premature luteinization after pituitary down- Kol et al. analysed the outcome of freeze-thaw cycles regulation the patient is given an estrogen preparation from with oocytes obtained with the use of six different doses of cycle day one onwards to mimic the proliferative phase.
ganirelix, in a multiple dose schedule. Even though there Estrogen may be administered as an oral preparation, skin was a negative effect of too high doses of ganirelix on patches, vaginal preparation or as subcutaneous implants implantation in the fresh cycle, there was a good pregnancy This is followed by concomitant administration of rate in subsequent freeze-thaw cycles. They concluded that progesterone to imitate the luteal phase. Progesterone may high dosages of ganirelix in the collecting cycles do not be administered as i.m. progesterone in oil injections or as adversely affect the potential of embryos to establish clinical tablets given orally or vaginally.
pregnancy in freeze-thaw cycles.
Lassale et al. claimed that GnRH agonist therapy In another retrospective study conducted by Byron et al.
adversely affects oocyte quality and freezing outcome, but they evaluated the outcome of frozen-thaw cycles with this could not be confirmed by others However, oocytes obtained either during a multiple dose protocol of recent studies showed that suppression with GnRH agonist cetrorelix, or after the use of a gonadotrophin-releasing for endometrial preparation is not necessary as pregnancy hormone (GnRH) agonist. A total of 101 subfertile couples and implantation rates are similar with or without GnRH a were included. These couples had a total of 222 transfers of down-regulation. Also, the procedure is simpler, less frozen-thawed pronuclear zygotes after IVF/intracytoplas- expensive and more convenient to the patient if performed mic sperm injection (ICSI) treatment. According to the without GnRH agonist stimulation protocol during various cycles, four groups were Embryo transfer of frozen thawed zygotes is performed after a twenty-four hour period of culture at cleavage stage.
Up to three cleaving embryos are transferred, according to 1. cetrorelix/recombinant FSH (recFSH) (69 cycles); the German Embryo Protection Law.
2. cetrorelix/human menopausal gonadotrophin (HMG) (10 Before transfer, attention should be paid to the degree of fragmentation and the regularity of blastomeres, each 3. GnRH-agonist/recFSH (71 cycles); and embryo being graded as 1, 2 or 3 (modified grading 4. GnRH-agonist/HMG (72 cycles).
according to Veeck, 1991) The grade of each embryo is multiplied by the number of blastomeres, to produce a The transfer cycles were mildly stimulated with trans- quality score. The total score of all embryos transferred is dermal estradiol. No statistically significant difference was accepted as the cumulative embryo score (CES) It is seen among the four groups regarding post-thaw survival important to clarify that this is the scoring system we follow, rate, cumulative embryo score, implantation rate and pre- as there are various ways to score the cleaved embryos.
Clinical pregnancies are defined by the presence of From all the previous studies, it could be concluded that positive fetal heartbeats. In these cases, the administration of frozen-thawed pronuclear zygotes obtained with the use of progesterone is continued up to week 12 of gestation.
GnRH antagonists give satisfactory implantation and pregnancy rates, similar to those obtained with a GnRH- agonist. These results do not depend on the gonadotrophins 5. The effect of stimulation protocol (HMG or recFSH) used in the collecting cycle.
It is known that ovarian stimulation protocols used in collection cycles may possibly be involved in the success of 6. Is there a difference between in vitro fertilization cryopreservation Furthermore, concerns have been (IVF) and intracytoplasmic sperm injection (ICSI)? raised recently about the possible impact of GnRH- antagonists on the quality of oocytes, embryo development Only few studies investigated the effect of cryopreserva- and implantation Although these questions are mainly tion on human embryos with perforated zonae. Al-Hasani et related to fresh cycles, the quality of oocytes may also affect al. compared cryopreservation of pronuclear stage human the outcome of freeze-thaw UNCORRECTED PROOF cycles. Consequently, these zygotes obtained either after classical in vitro fertilization or

Y. Orief et al. / Reviews in Gynaecological Practice xxx (2004) xxx–xxx after intracytoplasmic sperm injection After ICSI or [2] Lurie D, Check JH, Nazari A, Choe JK, Lee G. Cumulative pregnancy IVF, three fertilized oocytes from each patient were kept in rates after four embryo transfers of either fresh or frozen embryos. ClinExp Obstet Gynecol 2001;28:148–52.
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