Howard W.Jones Jr1,3, Henk J.Out2,. The overall troublesome aspects of presenting data from cryopreservation have been discussed previously (Jones.
Human Reproduction vol.12 no.7 pp.1522–1524, 1997
Cryopreservation: the practicalities of evaluation
Howard W.Jones Jr1,3, Henk J.Out2, Eric H.M.Hoomans2, Stefan G.A.J.Driessen2, and Herjan J.T.Coelingh Bennink2 lJones
Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, 601 Colley Avenue, Norfolk, VA 23507, USA, and 2NV Organon, PO Box 20, 5340 BH Oss, The Netherlands 3To
whom correspondence should be addressed
An attempt was made to integrate data from cryopreserved embryos with those from fresh embryos to obtain a realistic assessment of the role of cryopreservation in assisted reproductive treatment. Principles were applied to previously published data from a large prospective randomized multicentre study comprising recombinant and urinary follicle stimulating hormone in in-vitro fertilization. Key words: ART/cryopreserved embryos/FSH/IVF/multicentre prospective study
Introduction One of the main purposes in cryopreserving pre-zygotes/preembryos which are in excess after transferring an appropriate number of fresh pre-zygotes/pre-embryos is to realize the total pregnancy potential from the single egg harvest. In practical terms, this means that augmentation of the patient-specific pregnancy rate from cryopreservation would include only those pregnancies from cryopreserved material which occurred in patients who did not have a pregnancy from a fresh transfer. Therefore, pregnancies which occur from cryopreserved material among patients who had a pregnancy from freshly transferred material would not be counted as augmentation of the patient-specific pregnancy rate, but would be counted as multiple pregnancies from the same single egg harvest. A satisfactory and agreed-upon method of presenting the augmentation data has yet to be realized. For example, the American Society for Reproductive Medicine (ASRM), through its affiliate society, the Society for Assisted Reproductive Technology (SART), makes no attempt whatsoever to relate the cryopreservation results to the fresh results. It simply reports the fresh pregnancy rates separately from the cryopreservation pregnancy rates without association. In the UK, the Human Embryology and Fertilisation Authority (HFEA) counts a cryopreservation transfer as a cycle, and simply sums the pregnancies from fresh cycles with the number of pregnancies and transfers from cryopreservation, getting a combined figure that is not realistic and severely penalizes programmes with cryopreservation (Jones, 1996). 1522
The overall troublesome aspects of presenting data from cryopreservation have been discussed previously (Jones et al., 1995). The purpose of this paper is to pursue the reality of presenting data from cryopreservation utilizing the principles expounded above by applying previously published data from a large, prospective, randomized, assessor-blind, multicentre study comparing recombinant and urinary follicle stimulating hormone (FSH) in in-vitro fertilization cryopreservation (IVF) (Out et al., 1995). Materials and methods The various formulae to give a comprehensive evaluation of the supplementary effect of cryopreservation on a patient-specific basis were taken from a previous publication on this subject (Jones et al., 1995). In brief, for a comprehensive evaluation, four types of information were required: (i) the percentage of patients and percentage of pre-zygotes/pre-embryos with cryopreservation; (ii) the efficiency of the cryopreservation process; (iii) the pregnancy potential of surviving thawed pre-zygotes/pre-embryos; and (iv) the augmentation of the fresh pregnancy rate by the cryopreserved material. The clinical material applied to these formulae was taken from a large, prospective, randomized multicentre study comparing recombinant FSH (Puregon; NV Organon, Oss, The Netherlands) with urinary FSH (Metrodin; Organon) in IVF (Out et al., 1995). The clinical protocols were completely set forth in that publication. In brief, there were 19 centres with 585 recombinant FSH cycles and 396 with urinary human menopausal gonadotrophin (HMG) cycles. Patients were down-regulated with intranasal buserelin. The initial gonadotrophin dose was 2–3 ampoules per day and the dose was adjusted according to response. When at least three follicles were ù17 mm in diameter, 10 000 IU of human chorionic gonadotrophin (HCG) were given. A maximum of three fresh embryos was transferred. Cryopreservation data updated to March 1996 were obtained from the Organon database which collated the information from the 18 collaborative centres. The bulk of these cryopreservation data has not been previously published. The cryopreservation technique was carried out according to local protocols.
Results For complete evaluation, detailed information about four clusters of variables of the freeze/thaw process is required. Percentage of patients and percentage of pre-zygotes/preembryos with cryopreservation For a complete evaluation, it is necessary to know what percentage of the patients who have a fresh transfer also have material available for cryopreservation. For the same reason, it is also necessary to know the percentage of individual pre© European Society for Human Reproduction and Embryology
Cryopreservation: the practicalities of evaluation
Table I. Summary table of the number of subjects assigned to fresh embryo transfer, to cryopreservation and the cryopreservation rate
Table III. Summary table of the efficiency of cryopreservation expressed by the survival rate of thawed embryos
Overall
Overall
FSH treated subjects Subjects with fresh transfer Subjects with cryopreservationa Subjects fresh transfer rateb Subjects cryopreservation ratec
Treatment Puregon
Metrodin
585 500 216 85.5 43.2
396 329 129 83.1 39.2
FSH 5 follicle stimulating hormone. aNumber of subjects with frozen embryos, including eight subjects without a fresh transfer in the Puregon groups and four subjects without a fresh transfer in the Metrodin group. bNumber of subjects with fresh transfer/number of FSH-treated subjects. cNumber of subjects with cryopreservation/number of subjects with fresh transfer.
Table II. Summary table of the number of embryos assigned to fresh transfer and to cryopreservation Overall
FSH treated subjects Total no. embryos Embryos transferred fresh Embryos cryopreserved Embryo fresh transfer ratea Embryo cryopreservation rateb
Treatment Purgon
Metrodin
585 3897 1220 1259 31.3 32.3
396 2093 792 579 37.8 27.7
FSH 5 follicle stimulating hormone. aNumber of embryos transferred fresh/total number of embryos. bNumber of embryos cryopreserved/total number of embryos.
zygotes or pre-embryos which are assigned to fresh transfer and to cryopreservation. With this information, it is possible to evaluate the importance of pre-embryos not selected in both the fresh and cryopreservation transfer material. Utilizing the data mentioned, we found that 43.2% of patients treated with Puregon had sufficient material for fresh transfer as well as for cryopreservation, and the corresponding value for Metrodin was 39.2% (Table I). For the Puregon group, 31.3% of available pre-embryos were utilized for fresh transfer and 37.8% of available preembryos from the Metrodin-treated patients were utilized for this purpose (Table II). Additionally, for the Puregon group, 32.3% of the embryos were cryopreserved, and 27.7% for Metrodin. This means that 36.4% of the pre-embryos in the Puregon group were not selected for either fresh transfer or cryopreservation (i.e. deselected) and 34.5% in the Metrodin group were deselected (Table II). Efficiency of cryopreservation It is necessary to know the percentage of pre-zygotes/preembryos which survive the freezing process. The survival rate was 79.5% for the Puregon-treated group, and 81.8% for the Metrodin-treated group (Table III). Pregnancy potential of surviving pre-zygotes/pre-embryos There is a variety of ways of expressing the pregnancy potential of surviving pre-zygotes/pre-embryos, e.g. on a pre-zygotes/
Treatment
No. subjects Embryos cryopreserved Embryos thawed Surviving thawed embryos Embryo survival ratea aNumber
Puregon
Metrodin
216 1259 599 476 79.5
129 579 340 278 81.8
of surviving embryos thawed.
Table IV. Summary table of the pregnancy potential of surviving thawed embryos Overall
Treatment
Subjects with thawed transfers Total number of thawed transfers Subjects pregnant Total number of pregnancies Transfer specific pregnancy ratec Subject specific pregnancy rated aIncludes
Puregon
Metrodin
120a 153 22a 22 14.4 18.3
73 93 7b 7 7.5 9.6
three subjects who did not have a fresh transfer.
bIncludes one subject who did not have a fresh transfer. cTotal number of pregnancies/total number of thawed transfers. dNumber
of subjects pregnant/number of subjects with thawed transfers.
pre-embryo basis or on a per transfer basis regardless of the number transferred. In addition, the type of pregnancy can be defined, e.g. clinical pregnancies, ongoing pregnancies, term pregnancies, etc. In this example, clinical pregnancies per transfer is used. The transfer clinical pregnancy rate was 14.4% for the Puregon group and 7.5% for the Metrodin group (Table IV). Augmentation of the fresh pregnancy rate by cryopreservation The determination of augmentation by cryopreservation is the crux of the cryopreservation presentation concept. It is impossible to express the process entirely by a single formula. In this presentation, it is elected to define the pregnancy rate as clinical pregnancy rate, i.e. clinical pregnancies per transfer, and to limit the discussion to patients who have had adequate material for cryopreservation. Among the Puregon group, the overall cryo-augmented (interim) pregnancy rate is 38.9% and among the Metrodin group, 32.0%. The projected overall cryo-augmented (final) pregnancy rates are 49.5% and 35.7% respectively (Table V). It is to be noted that the projected (final) cryo-augmented pregnancy rate (by definition) includes pregnancies among as yet unthawed material on the assumption that the pregnancy rate among the unthawed material will be the same as it is among the thawed material. It is to be noted that the overall rate includes all cryopregnancies regardless of a previous fresh pregnancy. As indicated above, for a patient-specific rate, it is very important not to count as augmentation the pregnancies from cryopreservation among patients who have pregnancies by 1523
H.W.Jones et al.
Discussion Table V. Summary of the augmented and projected pregnancy rate; subjects with fresh transfer and cryopreservation Overall
Treatment Puregon
No. subjectsa 208 No. fresh pregnancies 62 No. thawed pregnancies 19 No. embryos cryopreserved 1155 No. embryos unthawed 618 No. embryos thawed 537 Pregnancy rate thawed embryosb (%) 3.5 c No. projected pregnancies 21.6 Augmented pregnancy rated (%) 38.9 Projected augmented pregnancy ratee (%) 49.5
Metrodin 125 34 6 549 239 310 1.9 4.5 32.0 35.7
aNumber
of subjects with fresh transfer and cryopreservation. Eight Puregon patients and four Metrodin patients who had cryopreservation without a fresh transfer have been excluded. bNo. pregnancies from thawed material expressed as a percentage of total number of embryos thawed in these patients. cPregnancy rate of thawed embryos3no. unthawed embryos. It is assumed that the pregnancy rate for the unthawed material will be the same as for the already thawed material. dNumber of fresh 1 thawed pregnancies/no. subjects with fresh transfer and cryopreservation. eAs above (d), including the projected number of pregnancies in the numerator.
Table VI. Summary table of the augmented and projected pregnancy rate compensating for the double counting of pregnancies in case a subject has more than one pregnancy Overall
Number of subjectsa Subjects with fresh or thawed pregnancies Number of embryos unthawedb Pregnancy rate thawed embryosc Projected number of pregnant subjectsd Augmented pregnancy ratee Projected augmented pregnancy ratef
Treatment Puregon
Metrodin
208 81 203 3.5 7.1 38.9 42.4
125 40 106 1.9 2.0 32.0 33.6
aNumber
of subjects with fresh transfer and cryopreservation. Eight Puregon patients and four Metrodin patients who had cryopreservation without a fresh transfer have been excluded. bNumber of embryos unthawed for subjects with no fresh or thawed pregnancy. cNumber of pregnancies from thawed transfers/number of thawed embryos. dProjected from the unthawed material among subjects without a fresh or thawed pregnancy, assuming that the pregnancy rate will be the same as for the already thawed material. eSubjects with fresh or thawed pregnancies/subjects with a fresh transfer and cryopreservation. fAs in e, including the projected number of pregnant subjects in the numerator.
fresh transfer or a previous cryothaw. By eliminating pregnancies of the type mentioned, the patient-specific cryo-augmented (interim) pregnancy rate can be recalculated. If this is done, the cryo-augmented (interim) pregnancy rates are 38.9% for the Puregon group and 32.0% for the Metrodin group, whereas the projected patient-specific cryo-augmented pregnancy rate becomes 42.4% for the Puregon group and 33.6% for the Metrodin group (Table VI). 1524
Only by the application of real numbers can there be a clear realization of the benefits of presenting cryopreserved data so that they reflect augmentation of the fresh pregnancies through the cryopreservation process. As indicated repeatedly above, it is the view of these authors that pregnancies resulting from the thawing of cryopreserved material among patients who have already had a fresh pregnancy from the same harvest or from a cryothaw from the same harvest should not be considered as augmenting the patient specific pregnancy rate, but rather should be considered as pregnancies which increase the multiple pregnancy rate (twins, triplets, etc.), although the birthdate of the twin or triplet, as the case may be, may be different. Thus, to achieve a patient-specific augmented pregnancy rate, it is extremely important in evaluating the role of cryopreservation to include as augmentation only those pregnancies from cryopreserved material among patients who did not have a pregnancy with a fresh transfer or a previous cryothaw from the same harvest. This represents true augmentation of the patient-specific expectation of pregnancy. In current practice where the number of fresh pre-embryos which can be transferred is limited by guidelines, official regulations, or whatever, it is essentially impossible to determine the merits of alternate protocols by the utilization of fresh pregnancy rates, for the simple reason that the ‘leftovers’ from fresh transfer can very well contain the critical information needed to evaluate the two alternative protocols. Furthermore, cryopreservation could and should become more and more important in assisted reproductive treatment (ART) because there is considerable pressure to reduce the incidence of multiple pregnancies per transfer (Jones, 1995; Salat-Baroux and Antoine, 1996). An obvious way to do this is to limit even more than now the number of pre-embryos transferred. This would result in more material for cryopreservation. Thus, it becomes even more important that the consequence of such a limitation be clearly and accurately expressed and recorded. It turns out in the examples cited that Metrodin and Puregon have many similar characteristics. In many categories, Puregon is at least equal to Metrodin. Acknowledgements The authors gratefully acknowledge the editorial assistance of Pauline M.Clynes, Editor, of the Jones Institute for Reproductive Medicine.
References Jones, H.W. Jr (1995) Twins or more. [Editor’s Corner]. Fertil. Steril., 63, 701–702. Jones, H.W. Jr, Veeck, L.L. and Muasher, S.J. (1995) Cryopreservation: the problem of evaluation. Hum. Reprod., 10, 2136–2138. Jones, H.W. Jr (1996) HFEA’s patient guidelines penalise the value of embryo preservation. Hum. Reprod., 11, 1364–1365. Out, H.J., Mannaerts, B.M.J.L., Driessen, S.G.A.J. and Coelingh Bennink, H.J.T. (1995) A prospective randomized, assessor-blind multicentre study comparing recombinant and urinary follicle stimulating hormone (Puregon versus Metrodin) in in-vitro fertilization. Hum. Reprod., 10, 2534–2540. Salat-Baroux, J. and Antoine, J.M. (1996) Multiple pregnancies: the price to pay. Eur. J. Obstet. Gynecol. Reprod. Biol., 65, S17–S18. Received on November 1, 1996; accepted on April 16, 1997
