High pregnancy rate after early human embryo freezing

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1 FERTLTY AND STERLTY Copyright The American Fertility Society Vol. 46 No.2 August 1986 Printed in U.SA. High pregnancy rate after early human embryo freezing Jacques Testart Ph.D.*t Bruno Lassalle* Joelle Belaisch-Allart M.D.:j: Andre Hazout M.D.:j: Robert Forman M.D. * Jean Daniel Rainhorn M.D.:j: Rene Frydman M.D.:j: Unite 187 nstitut National de la Sante et de la Recherche Medicale (lnserm) and H6pital Antoine Beclere Clamart France Human embryos produced by in vitro fertilization (NF) were frozen with l2-propanediol as a cryoprotectant. Embryo survival after thawing was related to the presence of a nucleus in frozen cells and decreased with the increasing number of cells in the frozen embryo. None of five embryos frozen 3 or 4 days after NF survived when thawed. Of48 early embryos (35 patients) frozen 1 or 2 days after VF 42 (87.5%) were transferred in 32 patients. Ten pregnancies were initiated after frozen embryo transfer (ET). f we exclude the three infertile patients who had sexual intercourse in the fertile period the pregnancy rate for each patient who had 1- or 2-day frozen embryo( s) was 22% (7 of 32). One of the pregnancies was obtained after ET of a 1-cell pronucleated frozen and thawed embryo. The rate of ongoing pregnancies after triple fresh ET was 23%. n patients having four embryos obtained in a single NF cycle the expected overall liveborn rate in an NF -ET program including embryo cryopreservation could theoretically equal that of natural human fertility. Fertil Steril 46: The current success rate of in vitro fertilization and embryo transfer (lvf-et) remains low despite considerable improvements in recent years. Many oocytes can be recovered from infertile patients and most of them fertilize and cleave underin vitro conditions. However failure of pregnancy after ET remains in the order of 70% to 80% indicating that embryo quality and/or stim- Received December ; revised and accepted April *Unite NSERM 187. treprint requests: Jacques Testart Ph.D. Unite NSERM 187 (Professor E. Papiernik) Hopital Antoine Beclere 157 rue de la Porte de Trivaux Clamart France. :j:service de Gynecologie-Obstetrique (Professeur E. Papier nik) Hopital Antoine Beclere. '268 Testart et a. Early human embryo freezing ulated patient receptivity to the embryo(s) act as unfavorable factors. Because it has been estimated that about 50% of human zygotes may have chromosomal anomalies in natural conception 1 inherent egg defects in the human may explain in part the low success rates after VF-ET. ET in natural cycles avoids changes in the reproductive tract induced in patients undergoing hormonal stimulation. There are several reasons to suppose that the practice of freezing and storing human. embryos produced by VFcould be of considerable value in improving the chances of pregnancy when numerous embryos are simultaneously obtained. n fresh mutiple-etthe chance for each embryo to develop decreases when the number of transferred embryos increases; i.e. the pregnancy rate after triple-et is only twice not three times Fertility and Sterility

2 that of single-et.2 Also we have demonstrated that there is twice the chance for an embryo to become a fetus when triple-et is performed in patients in whom five oocytes are recovered rather than following triple-et when only three 00cytes are recovered. 2 This observation demonstrates that embryos arising from high ovulatory patients are more able to initiate pregnancies than embryos from low ovulatory patients. Therefore embryo freezing could increase the chance of establishing a pregnancy by permitting the transfer over several cycles of embryos arising from a single oocyte recovery operation. Recent articles 3 4 have reported the success rate after transfer into the uterus of frozen and thawed human embryos. We have already proposed a freezing technique with 12-propanediol (PROH) as cryoprotector. 5 This article gives the first results obtained by transferring early human embryos frozen and thawed according to this technique. MATERALS AND METHODS OOCYTE RECOVERY Oocyte recovery was performed for the majority of patients during an VF cycle stimulated with clomiphene citrate (CC) (Clomid; Merrell Toraude Paris France) and human menopausal gonadotropin(nductor hmg; Searle Paris France) as previously described. 6 n addition in 15 cycles the oocyte recovery was programmed during a preliminary laparoscopy prior to inclusion in our VF program according to a technique recently described. 7 Briefly the cycle prior to the laparoscopy cycle was modified by the administration of norethisterone (Norluten Smith Kline & French Paris France) or an estrogen-progestagen contraceptive tablet (Triella Cilag Paris France) followed by a fixed schedule ovulation stimulation and induction therapy of CC hmg and human chorionic gonadotropin (hcg). nthis latter case hcg was systematically administered on a predetermined day and no hormonal or. ultrasonic monitoring of:follicular growth was performed. All the patients had been infertile for a minimum of 3 years. N VTRO FERTLZATON AND EMBRYO FREEZNG Oocytes were fertilized and cultured in Menezo'sB2 medium (Api-System La BalmeVol. 46;No. 2 August 1986 rpbs+20'hcs - o. 2 M SUCROSE +1.5M.pf()H M PROH Smln \ +0.1 M SUCROSE -0.5 M PROH -LT:""ST;';':RA:nW-=::::.m. Sm.n 2 C.mlf1 +1.0MPAOH embryo PBS.20\\hCS M SUCFllSE Sm.n SEEDNG O.JOe.mln "-l..rn. <L Figure 1 Schematic representation of the method used for freezing and thawing. les-grottes France) without any serum addition inside a modified neonatal incubator.8 When possible each patient received three embryos and supernumerary embryos were frozen between 1 'and 4 days after oocyte recovery.. n the 15 programmed cycles all the embryos obtained were frozen. Based: upon our previous observations5 cleaved embryos were selected for freezing on the basis of their cleavage stage (2-4- and 8-cell embryos were preferred over those in intermediate stages). Figure 1 summarizes the different steps of our freezing and thawing method. They are exactly the same as already described 5 except that sucrose was always added before freezing (0.1 M) and after thawing (0.2 M). FROZEN THAWED EMBRYO TRANSFER The patient returned to our unit 1 to 6 months after the freezing of their embryo(s). Daily plasma radioimmunoassay of luteinizing hormone (LH) allowed us to predict the day of ovulation. Daily LH assays were performed from the day when ultrasonography demonstrated a follicle of at least 15 mm. Three of 43 cycles were canceled. because there was. no LH surge at day 20 after the onset of menstruation. The LH surge was defined as when the LH concentration was at least twice that of the mean of the preceding days. Ovulation was estimated to occur the day following LH Testart et a. Early human embryo freezing 269

3 Table 1. Embryo Survival After Thawing According to the Day of Freezing Age of frozen embryos (days No. of frozen- No. of transafter in vitro thawed em- ferred embryos" % insemination) bryos" 1 16 (11) 14 (11) (24) 28 (21) (3) (2) anumbers in parentheses is the number of patients. surge detection. The embryos were transferred 1 to 4 days after ovulation according to their developmental age. ET took place between 0 and 3 hours after thawing except for four eggs which were cultured for 1 day from the pronucleated stage. All of the embryos preserving at least 50% of their initial number of cells were transferred. Two embryos with only one of four cells intact after thawing were transferred in two patients. Each of these embryos was simultaneously transferred with another better surviving embryo. Only one patient received three 3-cell embryos which resulted from the freezing of three embryos at the 4-7- and 9-cell stages. RESULTS None of 5 embryos frozen 3 or 4 days after oocyte recovery survived when thawed and 42 of 48 (87.5%) frozen at day 1 or 2 were transferred (Table 1). The developmental stage at the time of freezing influenced the survival rate of the embryos at thawing. The proportion of embryos keeping all their cells intact was 87% 33% 43% 12% and 0% for embryos frozen at the to 6- or 8- to 16-cell stages respectively (Table 2). The analysis of the results was only done for the earliest embryos i.e. those frozen at day 1 or 2. Of the embryos frozen at the pronucleate stage four were cultured for 1 day before transfer. Three of them cleaved to two cells (n = 2) or four cells (n = 1). n 24 embryos the presence of a nucleus in each cell was ascertained for all transferred embryos before freezing and after thawing. There was a relation between the presence of a nucleus in frozen embryo cells and the survival of these cells at thawing. Nucleated cells survived at a rate of 74% (40 of 54) compared with 20% (4 of 20) for anucleated cells at the time of freezing (P < 0.001). However two -cell embryos frozen after the disappearance of the pronuclei survived to freezing and thawing. Patients were transferred with one embryo (n = 23) two embryos (n = 8) or three embryos (n = 1); and there were six three and one pregnancies respectively. There was no difference in embryo survival or pregnancy rate after ET when oocytes came from monitored or programmed cycles. The rate of pregnancy tended to be higher when the patient received at least one cleaved embryo preserving its initial number of cells than when all transferred embryo(s) had only 50% to 80% of cells intact (60% versus 27% not significant Table 3). Ten of 32 transferred patients began a pregnancy (31 % Table 4). However most of the transferred patients (21 of 32) had damaged or patent tubes; there were embryos frozen during preliminary laparoscopy before the patients' acceptance into our VF-ET program. 7 There were twopregnancies in 11 patients with blocked tubes. Among the 21 patients with nonblocked tubes eight pregnancies occurred but only five could be proven to be due to the transfer of frozen embryo(s) because only these patients observed sexual abstinence during the fertile period. The other three patients who had been infertile for at least 3 years had intercourse 13 and 7 days before ET. The proportion of pregnancies definitely arising from ET was at least 21.9% (7 of 32 Table 4). When related to the patients undergoing freezing and thawing the pregnancy rate was between 21.9% and 28.6% depending upon whether or not sexual intercourse was considered. Three pregnancies (two of which are in patients with blocked tubes) are ongoing (more than 10 weeks) and three are normal uterine early clinical pregnan- Table 2. Embryo Survival After Thawing According to the Cleavage Stage at the Time of Freezing Cleavage stage ntact cells after thawing at freezing No. of frozen No. of trans- (no. of cells) embryos ferred embryos (%) (87.5%) 2 14 (87.5) (33.3%) (66.7) (42.9%) (85.7) (12.5%) (87.5) (0.0%) (20.0) 270 Testart et al. Early human embryo freezing Fertility and Sterility

4 Table 3. Proportion of Pregnant Patients According to the Rate of Cell Survival in 2 -Day Embryosa Cell survival for the best No. of trans surviving einbryos ferred embryos 100% % Total 1 3/7 2/7 5/14 2 3/3 0/3 3/ Proportion of 6/10 3/11 9/21 pregnant patients % af more than one embryo was transferred only the best surviving one was included in the table. cies. There was one tubal pregnancy two abortions and one biochemical pregnancy. All the clinical pregnancies were single pregnancies. f we consider the 45 frozen-thawed embryos for which pregnancy or pregnancy failure can be proven (32 patients) the chances for a frozen egg to become a clinically diagnosed pregnancy were 156% (7 of 45). n those patients in whom the embryos chosen for freezing were supernumerary the results of the direct transfer of their fresh embryos indicated that this prior selection did not affect their chances of pregnancy. n the 52 patients in whom supernumerary embryos were frozen there were 12 ongoing pregnancies at 10 weeks (23%) after the transfer of the three fresh embryos. Five of these pregnancies were twin pregnancies. DSCUSSON With PROH as cryoprotectant we were able to obtain a high pregnancy rate after the transfer of frozen-thawed human embryos. Our results compare favorably with those of others who used dimethylsulfoxide (DMSO)34 or glycerol.4 Whereas glycerol appeared adequate for freezini blastocysts4 and DMSO for freezing intermediate stages (2-3- and 4-day embryos)3 4 PROH gave favorable results only for very early human embryos (1 and 2 days). This could be an advantage in VF ET practice because there is no need for further culture before freezing the embryos being frozen at the time of or even before the transfer of their "siblings" in VF. t has been recently shown9 that PROH is more efficient than glycerol for freezing the human oocyte and we were able to obtain an ongoing pregnancy after the transfer of one frozen and thawed -cell embryo. Compared with other cryoprotectants (glycerol or DMSO) PROH seems to have a very low toxicity even when used in high concentrations.lo PROH penetrates easily inside human embryo blastomeres and restricts ice crystal formation. The time taken for the penetration and for the exit of cryoprotectant in human oocytes is longer for glycerol than for PROH. 9 The addition of sucrose which is a nonpermeable solute helps to obtain cell dehydration at room temperature before cooling. l1 t is supposed that with our protocol there is little or no formation of a lethal quantity of intracellular ice. The high survival rate observed for 1- or 4-cell embryos (87.5%) was confirmed by the in vitro cleavage of three of four -cell frozen and thawed embryos. The survival rate was inversely related to the number of cells in the frozen embryo as already reported. 5 This explains the exceedingly poor recovery of 3- or 4-day embryos with the use of our protocol. We also confirm our previous observations 5 that nucleated cells survive better than nonnucleated cells. t may be that osmotic variations or increasing electrolyte concentration induced by freezing has a more dramatic effect when nuclear material is free of membrane. However it was not possible to ascertain whether the nonnucleated cells were observed during mitotic division or whether they were degenerating. The proportion of pregnant patients after ET was much higher when cell survival in the embryo was 100% compared with 50% to 80%. t may be that the potential of early human embryos to re- Table 4. Pregnancy Occurrence After Transfer of 1- or 2-Day Frozen-Thawed Embryos According to Clinical Data Tubal function Blocked tubes Damaged or patent tubes All Transferred patients No. of pregnant patients Total With sexual abo stinence during fertile period 10 (31.2%) 5 No. of pregnancies definitely arising from frozen embryos (21.9%) Vol. 46 No.2 August 1986 Testart et al. Early human embryo freezing 271

generate after partial cellular destruction is less than that of other mammals in which artificial splitting of blastomere numbers does not have a marked effect on the subsequent ability to develop.

5 generate after partial cellular destruction is less than that of other mammals in which artificial splitting of blastomere numbers does not have a marked effect on the subsequent ability to develop.12 On the other hand there is a high frequency of monozygotic twinning in humans that demonstrates an ability to duplicate cell numbers and so it may be that our results are simply due to an intrinsic difference between in vitro fertilized embryos in their ability to survive freezing/thawing procedures. The pregnancy rate after frozen-thawed ET was very high (at least 21%) in spite of numerous transfers with only one embryo (23 of 32). This could be explained by transfer in the natural cycle and transferring embryos mainly from highly ovulatory patients.2 t can also be stated that crude assssment of ovulation time using LH assay only once a day during the ET cycle is adequate for determining the time of ET. There were not enough data for us to find a relation between the number of transferred embryos and occurrence of a pregnancy. The chances of a frozen and thawed embryo successfully developing until the stage at which it was ultrasonographically detectable was 15.6%. The rate of ongoing pregnancies for fresh embryos after triple transfers (23%) was comparable to the rate of pregnancies initiated for frozen embryos after single or double transfer. This means that there was not a deleterious effect of freezing and thawing on early human embryos. There was no difference in embryo survival or pregnancy rate when all embryos or only selected embryos were frozen. However freezing of all the embryos was only done in the 15 patients undergoing preliminary laparoscopy during a programmed cycle and no conclusion may be drawn. We also found that the choice of embryos for freezing at certain cleavage stages did not affect the pregnancy rate when the remaining fresh embryos were transferred. Twelve of the 52 patients (23%) undergoing embryo freezing were already pregnant after fresh ET and many of them (n = 5) have twin pregnancies. t may be anticipated that either several pregnancies may be obtained successively in the same patients or frozen embryos may be donated by the couples to other sterile patients. Our results show that natural human fertility may be at least equaled if four or five embryos are obtained from a single cycle of oocyte recovery. By freezing the one or two supernumerary embryos about a 10% liveborn rate could be anticipated after each single frozen ET. This could be added to the mini- 272 Testart et a. Early human embryo freezing mum 20% liveborn rate resulting from the triple fresh ET.1t is now necessary to establish the most effective policy for giving an infant to an infertile patient i.e. to determine the number of fresh embryos to be immediately transferred and the number to be frozen for later single transfer in natural cycles. Our opinion is that it may be realistic to reduce the number of transferred fresh embryos or even to cancel fresh ET in the VF cycle. ADDENDUM Two normal infants one boy and one girl have already been delivered. One of the infants was born after the transfer of a two pronuclei cyropreserved embryo. A total of 19 pregnancies after frozen embryo transfer (n = 70) have commenced from 86 VF cycles in which supernumerary fertilized eggs were obtained. REFERENCES 1. Schulman JD Dorfmann A Evans M: Genetic aspects of in vitro fertilization. Ann NY Acad Sci 442: Testart J Belaisch-Allart J Frydman R: Relationships between embryo transfer results and ovarian response and in vitro fertilization rate: analysis of 186 human pregnancies. Fertil Steril 45: Mohr LR Trounson AD Freemann L: Deep-freezing and transfer of human embryos. J n Vitro Fert Embryo Transfer 2: Fehilly CB Cohen J Simons RF Fishel SB Edwards RG: Cryopreservation of cleaving embryos and expanded blastocysts in the human: a comparative study. Fertil Steril 44: Lassalle B Testart J Renard JP: Human embryo features that influence the success of cryopreservation with the use of 12 propanediol. Fertil Steril 44: Belaisch-Allart J Hazout A Guillet-Rosso F Glissant M Testart J Frydmann R: Various techniques for oocyte recovery in an in vitro fertilization and embryo transfer program. J n Vitro Fert Embryo Transfer 2: Frydman R Rainhorn JD Forman R Belaisch-Allart J Hazout A Lassalle B Testart J: Pregnancies after programmed oocyte retrieval and embryo cryopreservation. Am J Obstet Gynecol. n press 8. Testart J Lassalle B Frydman R: Apparatus for the in vitro fertilization and culture of human oocytes. Fertil Steril 38: Bernard A moedemhe DA Shaw RW Fuller B: Effects of cryoprotectant on human oocyte. Lancet 1: Renard JP: The cryopreservation of mammalian embryos. n Human in Vitro Fertilization Actual Problems and Prospects Edited by J Testart R Frydman. Amsterdam Elsevier 1985 p Renard JP Bui-Xuan-Nguyen N Garnier V: Two-step freezing of two-cell rabbit embryos after partial dehydration at room temperature. J Reprod Fertil 71: Willadsen SM Polge C: Attempts to produce monozygotic quadruplets in cattle by blastomere separation. Vet Rec 108: Fertility and Sterility

Cryopreservation of cleaving embryos and expanded blastocysts in the human: a comparative study

FERTILITY AND STERILITY Copyright" 1985 The American Fertility Society Vol. 44, No.5, November 1985 Printed in U.s.A. Cryopreservation of cleaving embryos and expanded blastocysts in the human: a comparative