Abstract
The human mature oocyte is particularly sensitive to cooling and low temperatures in addition to freeze-thaw damage. The efficiency of oocyte cryopreservation including the pregnancy outcome is still low. The aim of our study is to briefly introduce our preliminary clinical results achieved with oocyte cryopreservation (CP). Our work focused on the use of a slow cooling procedure using the cryoprotectants propanediol (1.5 M) and sucrose (0.3 M). Following a short incubation of 4–6 hours thawed oocytes were injected with a single sperm (ICSI) and fertilization was assessed 12–16 hours later. Laser assisted hatching (LAH) was performed on all transferred embryos and embryo transfer (ET) was carried out 48–72 hours after ICSI. One-hundred and ten eggs were thawed and a survival rate of 76% (84/110) was obtained. Of the 84 oocytes which survived, 64 subsequently fertilized (64/84; 76%) following ICSI and on the following day 55 of those had cleaved (55/64; 86%). Fifty-two embryos were transferred in 29 patients (1.8 embryo/patient), and 7 (7/29; 24%) resulted in clinical pregnancy (1 twin pregnancy). One of the pregnancies encountered first trimester abortion (1/7; 14%). Implantation rate of 15.4% per embryo transferred (8/52) and 7.3% per egg thawed (8/110) were obtained. In all cases, chorion biopsy was performed and chromosomal anomalities were not detected. Our results provide further evidence that the procedure can be applied safely and with good success in clinical assisted reproduction. However, more work is needed since the survival and implantation rate should be improved.
| Abbreviations | ||
| CP | = | cryopreservation |
| ICSI | = | intracytoplasmic sperm injection |
| PrOH | = | propanediol |
| IVF | = | in vitro fertilization |
| AR | = | assisted reproduction |
| LAH | = | laser assisted hatching |
| ET | = | embryo transfer |
INTRODUCTION
The main biophysical factor of cellular disruption in the oocyte during cryopreservation (CP) is the extensive intracellular ice formation that can be avoided by an adequate cell dehydration process. The rate of movement of water out of the oocyte depends on the relative proportion of free water associated with macromolecules, membrane kinetics (composition and permeability characteristics of the cell membrane), temperature, the difference in osmotic pressure between the two sides of the membranes, and the surface area to volume ratio of the oocyte. Several data indicate that these properties are influenced and modified by many factors such as the developmental stage, maturity, age of oocyte, fertilization, and the species [Gardner et al. [Citation2007]; Leibo [Citation2008]; Fahy [Citation2007]; Fabbri [Citation2006]; Gook and Edgar [Citation2007]; Stachecki and Cohen [Citation2004]]. Hunter et al. [[Citation1992]] demonstrated that these properties also differed between individual mature (MII) oocytes.
Increasing the extent of dehydration of human oocytes prior to cooling was considered as a possible approach to improving the outcome of oocyte CP. The dehydration process can be affected by the presence of permeating and non-permeating cryoprotectants (CPA) in the freezing solutions (equilibration and loading solutions). The non-permeating CPAs increase the concentration of extracellular solutes generating an osmotic gradient across the cell membrane (intra- and extracellular environment), which draws water out of the cell causing the cell to dehydrate before the freezing procedure. The permeating CPAs also support the formation of an osmotic gradient, which limits shrinkage by replacing the lost water.
Survival rates were enhanced by increasing the concentration of the non-permeating sucrose from 0.1 M (39%) to 0.2 M (58%) or 0.3 M (83%) in the first report of this approach [Fabbri et al. [Citation2001]]. Extending the duration of dehydration in the 0.2 M sucrose further improved survival (70%). These modifications have continued to maintain higher survival rates of around 70% considering data pooled from clinical studies using 0.2 M sucrose [Yang et al. [Citation2002]; Porcu et al. [Citation1999]; Winslow et al. [Citation2001]; Bianchi [Citation2006]] and slightly higher (74%) using 0.3 M sucrose [Chen et al. [Citation2005]; Fosas et al. [Citation2003]; Li et al. [Citation2005]; Borini et al. [Citation2006b]; Chamayou et al. [Citation2006]; Levi Setti et al. [Citation2006]; De Santis et al. [Citation2007]; Barritt et al. [Citation2007]; Konc et al. [Citation2007]]. Higher fertilization and cleavage rates have been reported with 0.2 M when compared with 0.1 M sucrose [Chen et al. [Citation2004]]. Data show that significantly more embryos are generated when oocytes are cryopreserved in 0.2 M relative to 0.1 M sucrose. Similarly, survival (71 versus 24%), fertilization (80 versus 53%) and cleavage (91 versus 80%) rates were all significantly increased when oocytes were cryopreserved in 0.3 M relative to 0.1 M sucrose resulting in an overall 5-fold increase in available embryos [De Santis et al. [Citation2007]]. The fertilization (76%) and cleavage (94%) rates of oocytes cryopreserved in 0.2 M sucrose have been reported to be similar to the rates for fresh oocytes (80 and 97%, respectively) from the same clinic [Bianchi et al. [Citation2005a]; Bianchi [Citation2006]]. Similar fertilization (67 versus 67%) and cleavage (89 versus 98%) rates have also been reported for oocytes frozen in 0.3 M sucrose relative to fresh controls [Levi Setti et al. [Citation2006]]. Comparing the results of frozen oocytes cryopreserved in 0.2 M sucrose and fresh oocytes, Bianchi [[Citation2006]] reported similar implantation (13 versus 14%) and abortion (12 versus 10%) rates. However, the pooled data for all reports to date using 0.3 M sucrose show clearly that although the number of embryos generated is consistent with outcomes from the use of 0.2 M sucrose, relatively few of the resultant embryos (5%) implant. The data indicate that the implantation potential of embryos developed from frozen oocytes cryopreserved in 0.3 M sucrose is lower than that achieved with the use of 0.2 M sucrose and similar to that achieved with 0.1 M sucrose. Observations show that approximately a third of these implantations may abort [Bianchi et al. [Citation2005a]; Bianchi [Citation2006]; Borini et al. [Citation2006b]]. Results of others indicate that the majority of embryos originated from oocytes cryopreserved with 0.3 M sucrose show slowed development (64% at the 2-cell stage on day 2 and only 14% at the 4-cell stage) [Borini et al. 2006b; Bianchi et al. [Citation2005b]].
Concerns have been raised that an increase in the concentration of CPA, and hence in the extent of osmotic stress to which the oocyte is exposed, may jeopardize viability, with possible important consequences on the stability of the MII spindle [Pickering et al. [Citation1990]; Paynter et al. [Citation2005]]. Therefore, the aim of this study was to briefly introduce our preliminary results achieved with oocyte CP in our clinical ICSI+ET program. Our work focused on the use of a freezing procedure with medium containing 1.5 M propanediol (PrOH) plus an elevated concentration of sucrose (0.3 M) combined with a traditional slow-freezing protocol.
This paper reports the clinical outcome of mature oocyte cryopreservation using the slow freezing regimen with the cryoprotectants propanediol (PrOH, 1.5 M) and sucrose (0.3 M), together with the laser assisted hatching (LAH) of the subsequent embryo which develop.
Results
In 29 oocyte freeze-thaw cycles, 110 oocytes have been thawed resulting in a survival rate of 76% (84/110). Eighty-four oocytes were inseminated with ICSI and 64 (64/84; 76%) fertilized. In the group of fertilized oocytes we experienced a cleavage rate of 86% (55/64). Embryo transfers were performed in 29 patients (1.8 embryos per patient; 52 embryos). Seven clinical pregnancies as indicated by fetal heartbeat (24%) were achieved resulting in an implantation rate of 15.4% (8/52) per embryo and 7.3% (8/110) per egg thawed. In all cases, a chorion biopsy was performed and no chromosomal anomaly was detected. To date, five pregnancies completed successfully: 4 singleton with birth weights of 2800, 2800, 2700, 2800 g born at 36, 38, 38, 37 weeks of gestation with APGAR scores of 10, 10, 9, 10 and 1 set of twins with birth weights of 2700 and 2800 g and born at 38 weeks of gestation with APGAR scores of 9–10. There is one ongoing pregnancy (> 26 weeks) and one patient experienced spontaneous abortion at 10 weeks of pregnancy (1/7; 14%).
For comparison we have included results from a similar group of patients in which the oocytes (fresh) were collected at the same time as the frozen oocytes and all conditions were similar apart from embryos generated were not LAH prior to transfer. Data are presented in . There were no differences observed in the fertilization and cleavage rates (76 vs. 83%; 86 vs. 92%) between fresh and frozen. Although embryos which resulted from the frozen oocytes were LAH prior to embryo transfer and this was not performed on the embryos from fresh oocytes there was no difference in the pregnancy and implantation rates (24 vs. 33%; 15 vs. 18%). However, the results show a very pronounced difference in the cell stage on Day 2 of the resultant embryos between the frozen and fresh groups of oocytes (P < 0.05 measured by Chi square analysis). Our data show a slower embryo development in the frozen oocyte cycles relative to non-frozen/fresh cycles. In the frozen oocyte group 64% of the embryos remained in the 2-cell stage and only 17% were in the 4-cell stage on Day 2. In contrast, in the fresh group on Day 2 66% of embryos were already in 4-cell stage and only 25% of them were in the 2-cell stage. No difference was found in the miscarriage rates (14 vs. 16%).
Comparison of Fresh and Frozen Oocyte Outcomes
Discussion
To date the majority of successful pregnancies reported from mature oocyte CP involved the PrOH based slow freezing protocol with a different concentration of sucrose [Tucker et al. [Citation2004]; Porcu et al. [Citation2000]; Fabbri et al. [Citation2001]; Marina and Marina [Citation2003]; Borini et al. 2006a; Stachecki and Cohen [Citation2004]; Boldt et al. [Citation2006]; Chen et al. [Citation2005]; Gook and Edgar [Citation2007]; Bianchi [Citation2006]; Koutlaki-Kourti et al. [Citation2006]; De Santis et al. [Citation2007]; Chen et al. [Citation2004]]. Increasing the extent of dehydration of human oocytes prior to cooling was considered a possible approach to improving the outcome of oocyte CP.
Our data indicate and provide further evidence that slowly freezing oocytes in freezing solution containing 1.5 M PrOH and an elevated 0.3 M sucrose combined with ICSI at 4 hour post-thaw results in favourable outcome for survival, fertilization and cleavage. The overall survival, fertilization and cleavage rates are within the range of previous reports [Bianchi [Citation2006]; Borini et al. [Citation1998], 2006a; Fabbri et al. [Citation1998]; Tucker et al. [Citation1996], [Citation1998], [Citation2004]].
However, recent concerns have been raised connected with the use of 0.3 M sucrose during oocyte CP. Although survival, fertilization and cleavage rates appear normal, the resultant embryos exhibit a slower growth rate and the implantation is poor (∼5%), furthermore approximately a third of these implantations may abort [Borini et al. 2006b; Levi Setti et al. [Citation2006]; La Sala et al. [Citation2006]]. It can be speculated that the higher shrinkage caused by the elevated 0.3 M sucrose level may lead to cell injury responsible for decreased overall viability indicated by the poor implantation rate [Coticchio et al. [Citation2005], [Citation2006]]. The slower embryo development could be the likely reason for the poor implantation rate with the 0.3 M sucrose method [Bianchi et al. [Citation2005a]; Bianchi [Citation2006]]. The results of our study, similar to the previous observations, demonstrate that embryos from oocytes frozen in 0.3 sucrose exhibit slower development [Bianchi [Citation2006]; Bianchi et al. [Citation2005b]]. Comparing the embryo development between our non-frozen/fresh and frozen oocyte ICSI+ET cycles, a faster embryo growth was obtained in the fresh cycles.
In spite of the slower development, we obtained a higher implantation rate per transferred embryo (15.4 vs. ∼5%) and per oocyte thawed (7.3 vs. 2.4%) in our frozen oocyte cycles than suggested from the previously published literature [Bianchi [Citation2006]; Bianchi et al. [Citation2005b]; De Santis et al. [Citation2007]; Koutlaki-Kourti et al. [Citation2006]]. Comparing the implantation rates per transferred embryos between fresh and frozen oocyte cycles, we observed only a slightly lower result in the latter. The higher implantation rate that we obtained may be related to the LAH. The slower development of the embryos from oocytes frozen in 0.3 M sucrose might be associated with less cell cleavage and lower cell number (blastomer). The negative consequence of this phenomena may be that the blastocysts developing from frozen oocytes will not have sufficient cell number to become fully expanded blastocysts capable of rupturing the zona for hatching [Archer et al. [Citation2003]]. With LAH, we can assist the process of hatching and compensate the decreased hatching capability of these blastocysts. However, we have to take into consideration that the small number of patients and that patient selection may also be a reason for the improved results.
Despite numerous developments in IVF and ICSI the implantation rate of the replaced embryos remains low and it has been estimated that up to 85% of the replaced embryos do not implant. Assisted hatching (AH) has been proposed as a method for improving the capacity of the embryos to implant and several methods have been developed for AH. However, the results published by different clinics on the effectiveness of AH in promoting the implantation of embryos remain controversial. In a randomized double-blind controlled study Frydman et al. [[Citation2006]] found no improvement in IVF-ET outcome in woman aged >37 years after AH. However, Sallam et al. [[Citation2003]] in the context of a meta-analysis of randomized controlled trials determined that AH significantly increases pregnancy, implantation and ongoing pregnancy rates in patients with poor prognosis undergoing IVF or ICSI, particularly those with repeated failures. In our study, LAH was performed on all embryos originating from frozen oocytes prior to transfer. We decided to perform LAH, because CP of oocytes and embryos may lead to zona hardening, furthermore several data indicate that embryos from oocytes frozen with 0.3 M sucrose show slower embryo development that may compromise hatching and implantation. Thus AH has been advocated as a means of assisting the natural hatching process and enhancing implantation (see above).
There are very sensitive cellular structures in the oocyte that are extremely sensitive to low temperature and to the CPAs applied during CP [Cobo et al. [Citation2001]; Gook et al. [Citation1994]]. Therefore, one of the main concerns in cryopreserving oocytes is that the procedure may cause depolymerization of the meiotic spindle microtubules and other cytoskeletal elements leading to chromosomal abnormalities. However, in most surviving oocytes the spindle apparatus has the ability to properly reform upon thawing and to recover its functionality [Stachecki and Cohen [Citation2004]; Stachecki et al. [Citation2006]]. No chromosomal abnormalities to date have been detected even though depolymerization of the spindle resulting in cytogenic alterations was an earlier concern [Stachecki and Cohen [Citation2004]; Statechecki et al. 2004]. On the basis of 272 clinical pregnancies reported worldwide until July 2006, from embryos derived from cryopreserved human MII oocytes, Tur-Kaspa et al. [[Citation2007]] prepared the largest review of chromosomal studies of oocytes/embryos/infants and health status of children derived from frozen human MII oocytes. The rates of aneuploidy and malformations did not increase, and normal development was found in post-natal follow-up. They concluded that the data obtained reassured the safety of the clinical use of oocyte cryo-banking. Our data are in agreement with earlier observations of others of no difference in the miscarriage rates between frozen and fresh oocyte cycles (14 vs. 16%). Our results provide further evidence that despite the cellular structures and properties making the oocyte very sensitive to CP, the oocytes are freeze-able and can be safely cryopreserved. In all cases, a chorion biopsy was performed and chromosomal anomalies were absent. No differences were observed when the clinical parameters of the babies (birth weights, APGAR scores, duration of gestation, etc.) born from fresh or frozen oocyte were compared.
In summary, the results of oocyte CP remain inconclusive and the reported inconsistent survival and pregnancy rates obtained by both slow-freezing and vitrification indicate that more work is needed to find methods providing both excellent survival rates as well as maximal developmental competence for the thawed oocytes. However, great progress has been made in the procedure of oocyte CP and its efficacy has significantly improved. Data published in the literature indicate, that today 15 to 30 oocytes are needed to achieve one pregnancy, whereas in the past 100 to 150 were required. The results we achieved with oocyte CP provide further evidence that the procedure can be safely applied and with good success in clinical assisted reproduction. However, more work is needed since the survival and implantation rate can be improved. Additional trials are necessary to allow the evaluation of the two CP methods (slow freezing vs. vitrification) in terms of pregnancy achievement.
Materials and Methods
Only morphologically normal MII oocytes were selected for CP. The media used for freezing and thawing of oocytes were OocyteFreezeTM and OocyteThawTM (MediCult, Denmark). Equlibration with freezing medium based on PBS (Phosphate Buffered Saline) containing 1.5 M PrOH+0.3 M sucrose was performed at room temperature (OocyteFreezeTM, MediCult). Then oocytes were frozen in straws (3 oocytes per straw) in Planer III Kryo 10 cell freezer (Planer Products Ltd., Sunbury-on-Thames, UK). After seeding at minus 7°C, oocytes were slowly cooled (−0.3°C/min) to −30°C, than they were cooled with higher speed (−50°C/min) to −150°C before plunging into liquid nitrogen (LN2). After thawing, oocytes were rehydrated and PrOH was removed from the eggs by passage through 0.5 and 0.3 M sucrose in four steps (OocyteThawTM, MediCult). In vitro fertilization with ICSI was performed 4–6 hours after thawing, and fertilization was assessed 12–16 hours later. Assisted hatching with laser (LAH) was performed on all embryos prior to transfer procedure [Kanyo et al. 2004]. Embryo transfer was carried out 48–72 hours after ICSI at the 2 to 8 cell stages.
A comparison was made between fresh and frozen oocyte cycles carried out in the same period of time with patients attending our clinic with similar infertility history and being the same age.
The study had approval for patient materials and was approved by the local research and ethical committee.
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