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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.
culture for a further 24 h before embryo transfer. The surplus [3] Schnorr JA, Doviak MJ, Muasher SJ, Jones Jr HW. Impact of a zygotes were cryopreserved using the 'open freezing cryopreservation program on the multiple pregnancy rate associated system' and 1,2-propanediol and sucrose as cryoprotectants.
with assisted reproductive technologies. Fertil Steril 2001;75:147–51.
A cohort of 817 and 1626 zygotes in pronuclear stage were [4] Tiitinen A, Halttunen M, Harkki P, Vuoristo P, Hyden-Granskog C.
frozen after IVF and ICSI, respectively. Of these, 333 and Elective single embryo transfer: the value of cryopreservation. HumReprod 2001;16:1140–4.
744 zygotes have been thawed, of which 78 and 76.5% were [5] Tiitinen A, Husa LM, Tulppala M, Simberg N, Seppala M. The effect morphologically intact zygotes after IVF and ICSI of cryopreservation in prevention of ovarian hyperstimulation syn- respectively. From the 204 (ICSI) and 89 (IVF) zygote drome. Br J Obstet Gynecol 1995;102:326–9.
transfers performed, 34 (17%) and 18 (20%) pregnancies [6] Damario MA, Hammit DG, Galantis BA, et al. Pronuclear stage were established. Both groups showed a similar abortion rate cryopreservation after intracytoplasmic sperm injection and conven-tional IVF: implications for timing of the freeze. Fertil Steril of approximately 20%. They concluded that pronuclear stage zygotes resulting from ICSI can be successfully [7] Al-Hasani S, Demirel LC, Scho¨pper B, et al. Pregnancies achieved frozen/thawed and the survival and pregnancy rates achieved after frozen–thawed pronuclear oocytes obtained by intracytoplasmic are comparable to those for zygotes obtained after IVF.
sperm injection with spermatozoa extracted from frozen–thawed Aytoz et al. compared the obstetric outcome and the testicular tissues from non-obstructive azoospermic men. Hum Reprod1999;14:2031–5.
pregnancies after the transfer of fresh and cryopreserved [8] Veeck LL, Amundson CH, Brothman LJ, et al. Significantly enhanced embryos obtained by ICSI. They found a lower implantation pregnancy rates per cycle through cryopreservation and thaw of rate in the frozen ICSI group, indicating a decreased vitality pronuclear stage oocytes. Fertil Steril 1993;59:1202–7.
of the cryopreserved ICSI embryos. Furthermore, they [9] Van den Abbeel E, Camus M, Van Waesberghe L, et al. A randomized reported a higher miscarriage rate in the frozen ICSI group.
comparison of the cryopreservation of one cell human embryos with aslow controlled-rate cooling procedure or a rapid cooling procedure by direct plunging into liquid nitrogen. Hum Reprod 1997;12:1554–60.
[10] Trounson AO, Peura A, Kirby C. Ultrarapid freezing: a new low cost and effective method of embryo cryopreservation. Fertil Steril Cryopreservation of human embryos has been introduced [11] Gordts S, Roziers P, Campo R, Noto V. Survival and pregnancy into clinical IVF in order to preserve supernumerary outcome after ultrarapid freezing of human embryos. Fertil Steril1990;53:469–72.
embryos for a later transfer. Human embryos at different [12] Feichtinger W, Hochfellner C, Ferstl U. Clinical experience with ultra- developmental stages have been frozen with variable success rapid freezing of embryos. Hum Reprod 1991;6:735–6.
rates. The pronuclear stage appears to be the optimal stage [13] Park SP, Kim EY, Oh JH, Nam HK, Lee KS, Park SY, et al. Ultra-rapid freezing of human multipronuclear zygotes using electron microscope Several protocols of freezing have been formulated for grids. Hum Reprod 2000;15:1787–90.
[14] Liebermann J, Tucker MJ, Graham JR, Han T, Davis A, Levy MJ.
cryopreservation of human pronuclear zygotes among which Blastocyst development after vitrification of multipronuclear zygotes the conventional (slow) freezing has been the most widely using the Flexipet denuding pipette. RBM Online 2002;4:148–52.
used method of storage up to the present.
[15] Selman HA, El-Danasouri I. Pregnancies derived from vitrified human Other methods for freezing of the pronuclear zygotes zygotes. Fertil Steril 2002;77:422–3.
have been postulated like ultra-rapid freezing technique and [16] Van der Auwera I, Cornillie F, Ongkowidjojo R, Pijnenborg R, Koninckx PR. Cryopreservation of pronucleate mouse ova: slow versus ultra-rapid freezing. Hum Reprod 1990;5:619–21.
The refinement of vitrification in the last few years leads [17] Shaw JM, Diotallevi L, Trounson AO. A simple rapid 4.5 M dimethyl- this technique to be the method of choice for cryopreservation sulfoxide freezing technique for the cryopreservation of one-cell to of human zygotes and all stages of embryonic development.
blastocyst stage preimplantation mouse embryos. Reprod Fertil Dev This conclusion can be drawn because of the high success [18] Nowshari MA, Nayudu PL, Hodges JK. Effect of cryoprotectants and rates after thawing in these different stages as well as the their concentration on post-thaw survival and development of rapid simplicity of the procedure and the economic advantages.
frozen–thawed pronuclear stage mouse embryos. Hum Reprod 1995; For future research it will be important to establish a cryopreservation regimen which allows survival not only for [19] Bagis H, Odaman H, Sagorkaya H, Dinnyes A. Production of trans- the zygotes but also of the associated somatic cell genic mice from vitrified pronuclear stage embryos. Mol Reprod Dev2002;61:173–9.
components of the tissues.
[20] Hochi S, Hirabayashi M, Hirao M, Kato M, Kobayashi T, Kimura K, et al. Effect of cryopreservation of pronuclear-stage rabbit zygotes on the morphological survival, blastocyst formation, and full-term develop- ment after DNA microinjection. Mol Reprod Dev 2001;60:227–32.
[21] Al-Hasani S, Ludwig M, Karabulut O, et al. Results of intracytoplas- [1] Bergh C, Josefsson B, Nilsson L, Hamberger L. The success rate in a mic sperm injection (ICSI) using microprocessor controlled Transfer- Swedish in-vitro fertilization unit: a cohort study. Acta Obstet Gynecol Man Eppendorf Manipulator system. J Middle East Fertil Soc UNCORRECTED PROOF

Y. Orief et al. / Reviews in Gynaecological Practice xxx (2004) xxx–xxx [22] Ludwig M, Scho¨pper B, Al-Hasani S, Diedrich K. Clinical use of a [37] Simon A, Hurwitz A, Zentner BS. Transfer of frozen thawed embryos pronuclear stage score following intracytoplasmic sperm injection: in artificially prepared cycles with and without prior gonadotrophic impact on pregnancy rates under the conditions of the German embryo releasing hormone agonist suppression: a prospective randomized protection law. Hum Reprod 2000;15(2):325–9. February.
study. Hum Reprod 1998;18:2712–7.
[23] Al-Hasani S, Ludwig M, Gagsteiger F, et al. Comparison of cryopre- [38] Veeck LL. Preembryo grading. In: Brown CL., Vaughn VM., Lump- servation of supernumerary pronuclear human oocytes obtained after kin K, editors. Atlas of the Human Oocyte and Early Concepts.
intracytoplasmic sperm injection (ICSI) and after conventional in- Baltimore: Williams and Wilkins; 1991. p. 121–31.
vitro fertilization. Hum Reprod 1996;11:604–7.
[39] Steer CV, Mills CL, Tan SL, et al. The cumulative embryo score: a [24] Lassalle B, Testart J, Renard J. Human embryo features that influence predictive embryo scoring technique to select the optimal number of the success of cryopreservation with the use of 12 propanediol. Fertil embryos to transfer in an IVF-ET programme. Hum Reprod [25] Fahy GM, MacFarlane DR, Angell CA, Meryman HT. Vitrification as [40] Van Steirteghem AC, Van den Abbeel E, Smitz J, et al. Cryopreserva- an approach to cryopreservation. Cryobiology 1984;21:407–26.
tion of supernumerary embryos after GIFT and IVF in clomid-HMG [26] Liebermann J, Tucker MJ. Effect of carrier system on the yield of and buserelin-HMG stimulated cycles. In: Abstract Book of the VI human oocytes and embryos as assessed by survival and develop- World Congress on In Vitro Fertilization and Alternate Assisted mental potential after vitrification. Reproduction 2002;124:483–9.
Reproduction. Abstract no. 52. Jerusalem, Israel, 2–7 April, 1989.
[27] Jelinkova L, Selman HA, Arav A, Strehler E, Reeka N, Sterzik K. Twin [41] Van den Abbeel E, Smitz J, Camus M, et al. Evaluation of fresh and pregnancy after vitrification of 2-pronuclei human embryos. Fertil cryopreserved supernumerary embryos after clomiphene-HMG and buserelin-HMG stimulated GIFT cycles. In: Abstract Book 2nd Joint [28] Van den Abddel E, Camus M, Van Waesberghe L, Devroey P, Van ESCO-ESHRE Meeting. Abstract no 82. Milano, Italy, August 29– Steirteghem AC. Viability of partially damaged human embryos after September 1, 1990. p. 26–7, cryopreservation. Hum Reprod 1997;12:2006–10.
[42] Van der Elst J, Van den Abbeel E, Camus M, et al. Long-term [29] Uechi H, Tsutsumi O, Morita Y, Takai Y, Taketani Y. Comparison of evaluation of implantation of fresh and cryopreserved human embryos the effect of controlled rate cryopreservation and vitrification on 2 cell following ovarian stimulation with buserelin acetate-human meno- mouse embryos and their subsequent development. Hum Reprod pausal gonadotrophin (HMG) or clomiphene citrate-HMG. Hum [30] American Society for Reproductive Medicine, Society of Assisted [43] Hernandez ER. Embryo implantation and GnRH antagonists: embryo Reproductive Technology. Assisted reproductive technology in the implantation: the Rubicon for GnRH antagonists. Hum Reprod United states: 1996 results generated from theAmerican Society for Reproductive Medicine, Society of Assisted Reproductive Technology [44] Nikolettos N, Al-Hasani S, Felberbaum R, et al. Comparison of Registery Fertil Steril 1999;71:798–806.
cryopreservation outcome with human pronuclear stage oocytes [31] Younis AI, Toner M, Albertini F, Biggers JD. Cryobiology of non obtained by the GnRH antagonist, cetrorelix, and GnRH agonists.
human primate oocytes. Hum Reprod 1996;11:156–65.
Eur J Obstet Gynaecol Reprod Biol 2000;93:91–5.
[32] Be Nun I, Shulman A. Induction of artificial endometrial cycles with [45] Kol S, Lightman A, Hillensjo T, et al. High doses of gonadotrophin- s.c. oestrogen implants and injectable progesterone in in vitro ferti- releasing hormone antagonist in in-vitro fertilization cycles do not lization treatent with donated oocytes: a preliminary report. Hum adversely affect the outcome of subsequent freeze-thaw cycles. Hum [33] Lassale B, Testart J, Renard JB. Human embryo features that influence [46] Byron A, Nikos N, Safaa A-H. Proceedings of ESHRE Campus the success of cryopreservation with the use of 1,2propanediol. Fertil Workshop: GnRH antagonists: from basic science to clinical applica- tion: Outcome of cryopreserved pronuclear oocytes obtained after [34] Navot D, Rosenwaks Z, Anderson F, Hodgen GD. Gonadotrophin ovarian stimulation with either HMG or recFSH and the GnRH- releasing hormone induced ovarian hyperstimulation: low dose side antagonist cetrorelix. Reprod Biomed Online 2002;5(Suppl. 1).
effects in women and monkeys. Fertil Steril 1991;55:1069–75.
[47] al-Hasani S, Ludwig M, Gagsteiger F, Kupker W, Sturm R, Yilmaz A, [35] Demoulin A, Jouan C, Gerday C, Dubois M. Pregnancy rates after et al. Comparison of cryopreservation of supernumerary pronuclear transfer of embryos obtained from different stimulation protocols and human oocytes obtained after intracytoplasmic sperm injection (ICSI) frozen at either pronucleate or multicellular stages. Hum Reprod and after convention al in-vitro fertilization. Hum Reprod 1996; [36] Queenan JT, Ramey JW, Seltman HJ. Transfere of cryopreserved [48] Ayotz A, Van del Abbel E, Bounduelle M. Obstetric outcome of thawed pre-embryos in a cycle using exogenous steroids without prior pregnancies after the transfer of cryopreserved and fresh embryos gonadotropin releasing hormone agonist suppression yields favourable obtained by conventional in vitro fertilization and intracytoplasmic pregnancy results. Hum Reprod 1997;12:1176–80.
sperm injection. Hum Reprod 1999;14:2619–24.



CSIRO PUBLISHING Historical Records of Australian Science, 2013, 24, 242– Mollie Elizabeth Holman 1930–2010 Elspeth M. McLachlanA,C and G. David S. HirstB A Neuroscience Research Australia, Randwick, NSW 2031, Australia. B86 Caroline Street, South Yarra, Vic. 3141, Australia. CCorresponding author. Ema Mollie Holman was a biophysicist whose work on the autonomic nervous system and the innervation of smooth muscle was seminal in advancing knowledge of its behaviour at a cellular level. She was particularly known for her technical expertise in microelectrode recording of membrane potential from single smooth muscle cells, and the interpretation of their electrical activity, both spontaneous and in response to transmitters released from their autonomic nerves.


Prof. Francesco Castelli Clinica di Malattie Infettive e Tropicali Centro Interuniversitario Ricerca sulla Malaria (CIRM) Università di Brescia 2° U.O. di Malattie Infettive Azienda Ospedaliera Spedali Civili di Brescia WHO Collaborating Center for TB/HIV co-infection Malaria: nuove prospettive terapeutiche Conflicts of interest