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Freeze-all, oocyte vitrification, or fresh embryo transfer? Lessons from an egg-sharing donation program
ORIGINAL ARTICLE: ASSISTED REPRODUCTION 1 2 3 Q1 4 5 6 7 8 9 10 a,b,c Amanda Souza Setti, M.Sc.,a,c Q4 Daniela Paes Almeida Ferreira Braga, D.V.M., Ph.D., 11 ssia Sa vio Figueira, Ph.D.,a Matheus de Castro Azevedo, B.Sc.,a Assumpto Iaconelli Jr., M.D.,a Rita Ca 12 ~es Lo Turco, D.V.M., Ph.D.,b and Edson Borges Jr., M.D., Ph.D.a,c Q2 Edson Guimara 13 a ~ o Humana, Departamento de Cirurgia, Universidade Fertility–Medical Group; b Disciplina de Urologia, Area de Reproduc¸a 14 ~o Humana Assistida, Sa ~ o Paulo, ~o Paulo; and c Instituto Sapientiae–Centro de Estudos e Pesquisa em Reproduc¸a Federal de Sa 15 Brazil 16 17 18 19 Objective: To compare the outcomes of ETs using cryopreserved embryos, cryopreserved oocytes, or fresh embryos. 20 Design: Observational, cohort study. 21 Setting: Private university-affiliated fertility center. 22 Patient(s): This study included 8,210 mature oocytes obtained from 425 oocyte donors. Of those, 5,440 were used for the donors' own cycles (Fresh Oocyte Cycles Group), and 2,770 were cryobanked for 425 recipients (Banked Donor Egg Group). All of the oocytes were 23 sperm injected, resulting in 4,585 embryos from the donors' own cycles and 2,128 embryos from the recipients’ cycles. For the donor 24 cycles, embryos were either cryopreserved and transferred during a subsequent cycle (Thaw Cycles Group, 3,209 embryos), or they were 25 transferred during a fresh cycle (Fresh Cycles Group, 1,307 embryos). For the recipient cycles, embryos derived from vitrified oocytes 26 were transferred (Vitrified Oocytes Group, n ¼ 425 cycles, 2,128 embryos). 27 Intervention(s): Oocyte/embryo vitrification and intracytoplasmic sperm injection. 28 Main Outcome Measure(s): Embryo quality, pregnancy, and implantation rates. 29 Result(s): Decreased embryo quality and lower rates of blastocyst formation were observed among embryos derived from vitrified oocytes. The highest pregnancy and implantation rates were noted for the Thaw Cycles Group, followed by the Banked Donor Egg Group; 30 the Fresh Cycles Group had the lowest rates. 31 Conclusion(s): Oocyte vitrification followed by intracytoplasmic sperm injection leads to lower embryo developmental competence 32 compared with when fresh insemination methods are used. However, pregnancy and implantation rates are higher when embryos 33 are transferred into a ‘‘more receptive’’ endometrium, free of the adverse effects of gonado34 Use your smartphone tropin. Moreover, the freeze-all method leads to exceptional clinical outcomes. (Fertil SterilÒ to scan this QR code 35 2016;-:-–-. Ó2016 by American Society for Reproductive Medicine.) and connect to the Key Words: Cryobanking, embryo cryopreservation, freeze-all, oocyte vitrification 36 discussion forum for 37 this article now.* Discuss: You can discuss this article with its authors and with other ASRM members at http:// 38 fertstertforum.com/bragad-embryo-oocyte-vitrification/ * Download a free QR code scanner by searching for “QR 39 scanner” in your smartphone’s app store or app marketplace. 40 41 42 ince the development of cryocollection cycle. Cryopreservation elimwish to postpone maternity, the cryo43 preservation, this technique has inates the need to transfer multiple empreservation of oocytes has become 44 been a vital instrument of bryos, thereby significantly reducing more and more popular and has found 45 increasing importance in assisted reprothe rate of multiple pregnancies (2–4). other applications in areas such as 46 ductive technology (ART) (1). The cryoDriven by an increasing interest in oocyte donation (5). Oocyte donation, 47 preservation of cleavage-stage preserving the fertility potential of in which the oocyte and subsequent 48 embryos ensures the best use of the young women undergoing gonadoembryo qualities are optimized by oo49 fertility potential of an oocytetoxic treatments or even those who cytes donated from young women, is 50 quite a well-established treatment for 51 female infertility (6). With the introducReceived January 20, 2016; revised April 8, 2016; accepted May 9, 2016. 52 D.P.A.F.B. has nothing to disclose. A.S.S. has nothing to disclose. R.C.S.F. has nothing to disclose. tion of vitrification, it is now possible to 53 M.d.C.A. has nothing to disclose. A.I. has nothing to disclose. E.G.L.T. has nothing to disclose. vitrify and warm unfertilized eggs at E.B. has nothing to disclose. 54 Reprint requests: Edson Borges, Jr., M.D., Ph.D., Fertility–Medical Group, Scientific Research, Av. Brinear maximal efficiency, resulting in 55 ^ nio 4545, Sa ~o Paulo, Sa ~o Paulo 01401-002, Brazil (E-mail: edson@fertility. gadeiro Luis Anto high cell survival rates (7). The use of 56 com.br). frozen donor eggs avoids some diffi57 Fertility and Sterility® Vol. -, No. -, - 2016 0015-0282/$36.00 culties derived from the use of fresh 58 Copyright ©2016 American Society for Reproductive Medicine, Published by Elsevier Inc. oocyte donations, such as the http://dx.doi.org/10.1016/j.fertnstert.2016.05.004 59
Freeze-all, oocyte vitrification, or fresh embryo transfer? Lessons from an egg-sharing donation program
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synchronization between the donor cycle and the recipient endometrium (8). In addition, the use of donor oocyte cryobanking may provide patients with more choices when selecting oocytes, more flexibility in timing pregnancy, and potential reductions in the cost of treatments (1, 9). It has been reported that controlled ovarian stimulation (COS) is associated with impaired endometrial receptivity in IVF cycles (10–13). This may be explained by an embryo– endometrium asynchrony: there have been reports of a positive correlation between embryo developmental pace and implantation in fresh autologous IVF cycles. This asynchrony has not, however, been detected in oocyte donation cycles or cycles using frozen–thawed embryos (14, 15). In frozen–thawed cycles, when COS is not performed, pregnancy rates have been reported to be higher than in fresh autologous cycles (16). Considering the adverse effects of COS on endometrial receptivity, the freeze-all policy has emerged as an alternative to fresh embryo transfer to improve IVF outcomes (17, 18). With the freeze-all policy, an entire cohort of embryos is cryopreserved to be transferred later during a natural cycle, or during a cycle with hormonal replacement for endometrial priming. This provides a more physiologic environment for ET (18). It has previously been suggested that oocyte cryopreservation could result in disruption of the oolema molecular machinery required for normal segregation of chromosomes (19, 20). However, other studies have suggested that embryo quality is not affected by the oocyte vitrification procedure. Similar to embryos derived from fresh oocytes, it has been shown that vitrified oocytes preserve the potential to be fertilized and to develop into high-quality blastocysts (21). Moreover, in a paired randomized, controlled trial, Forman et al (22) have demonstrated that oocyte vitrification does not increase the rate of aneuploidy or diminish the implantation potential of viable blastocysts. Likewise, embryos can be consistently cryopreserved and warmed to yield pregnancy outcomes similar to those achieved after the use of fresh embryos (23). To date, however, little is known about the optimal oocyte/embryo stage that should be used for vitrification. Although it has been strongly suggested that ET during a natural or endometrium-prepared cycle is a better approach than transfer during a COS cycle, the question about the vitrification of oocytes or embryos remains under debate. Therefore, the goal of the present study was to compare the outcomes of ETs using cryopreserved embryos or cryopreserved oocytes in endometrial primed cycles by making use of well-controlled oocyte sampling (i.e., derived from oocyte donor patients). A secondary objective was to compare the quality of embryos derived from fresh or vitrified oocytes. Last, this study aimed to confirm the superiority of the freezeall protocol over fresh embryo transfer during COS cycles.
MATERIALS AND METHODS Study Design This study was performed in a privately assisted fertilization center in Brazil and included 8,210 viable mature oocytes obtained from 425 oocyte donors: 5,440 were used for the
donors’ own cycles (Fresh Oocyte Cycles Group) and 2,770 were cryobanked for 425 oocyte recipients (Banked Donor Egg Group). After warming, 2,635 oocytes survived. All oocytes were submitted to sperm injection, resulting in 4,585 embryos obtained from the Fresh Oocyte Cycles Group and 2,128 embryos obtained from the Banked Donor Egg Group. Concerning the Fresh Oocyte Cycles Group, the embryos were either cryopreserved and transferred in a subsequent cycle (Thaw Cycles Group, n ¼ 297 cycles, 3,209 embryos) or transferred during a fresh cycle (Fresh Cycles Group, n ¼ 128 cycles, 1,307 embryos). Therefore the Fresh Oocyte Cycles Group was split into two other groups: the Thaw Cycles Group and the Fresh Cycles Group (Fig. 1A). For the recipient cycles, embryos derived from vitrified oocytes were transferred (Banked Donor Egg Group, n ¼ 425 cycles, 2,128 embryos; Fig. 1A). All of the embryos were evaluated 16–18 hours after intracytoplasmic sperm injection (ICSI) on days 2, 3, and 5 of development when ET was performed. To evaluate the efficiency of the use of fresh or vitrified oocytes, the groups Fresh Oocyte Cycles and Banked Donor Egg Group were compared for fertilization rate, embryo quality on days 2 and 3, and blastocyst formation rate (Fig. 1B). The oocyte/embryo survival rate, total usable embryos rate, implantation rate, pregnancy rate, and miscarriage rate were compared among the Thaw Cycles Group, Fresh Cycles Group, and Banked Donor Egg Group (Fig. 1B). The oocyte/embryo survival rate was calculated by the number of surviving oocytes or embryos after warming divided by the number of vitrified oocytes or embryos. Total usable embryos was calculated by the number of transferred embryo plus the number of supernumerary cryopreserved embryos for future use dived by the total number of obtained embryos. The implantation rate was defined as the total number of gestational sacs divided by the total number of embryos that were transferred. Clinical pregnancy was defined as the presence of a gestational sac that could be visualized using ultrasound 4–6 weeks after ET, and miscarriage was defined as pregnancy with a total loss of gestational sacs before 20 weeks’ gestation. All of the cases with severe spermatogenic alterations, including frozen and surgically retrieved sperm, were excluded from the study. Written informed consent, in which patients agreed to share the outcomes of their cycles for research purposes, was obtained; the study was approved by the local institutional review board.
Controlled Ovarian Stimulation and Laboratory Procedures Controlled ovarian stimulation was achieved by pituitary blockage using a GnRH antagonist (Cetrotide, Serono); ovarian stimulation was performed using recombinant FSH (Gonal-F, Serono). Follicular growth was monitored using transvaginal ultrasound examination starting on day 4 of gonadotropin administration. When adequate follicular growth and serum E2 levels were observed, recombinant hCG VOL. - NO. - / - 2016
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Fertility and Sterility® 237 FIGURE 1 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 Q5 Description of experimental groups and comparisons. 285 Almeida Ferreira Braga. Embryo or oocyte vitrification? Fertil Steril 2016. 286 287 288 289 (Ovidrel, Serono) was administered to trigger the final 290 follicular maturation. The oocytes were collected 35 hours 291 after hCG administration through transvaginal ultrasound 292 ovum pick-up. 293 The recovered oocytes were assessed to determine their 294 nuclear status, and those in metaphase II were submitted to 295 ICSI following routine procedures (24).
A
B
Endometrial Preparation After menses, endometrial development was followed by ultrasound examination, and the patients receive 200 mg of transdermal 17b-E2 every 3 days (Estradot, Noven Pharmaceuticals). Approximately 14 days after initiation of E2 administration, serum E2 levels and endometrial thickness
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were determined. When endometrium showed proliferative morphology and thickness of at least 7.5 mm, 600 mg of P was vaginally administered per day. The administration of P was divided into 3 doses that were started (Utrogestan, Farmoquímica) and maintained until ET. Both 17b-E2 and P were administered concomitantly for 10 days after ET. The 17b-E2 and P treatments were suspended in the presence of a negative b-hCG test. In the presence of a positive bhCG test, the 17b-E2 and P treatments were maintained until weeks 6 and 12 of gestation, respectively. For COS cycles and fresh embryo transfers, on the day of ovum pick-up, patients received 600 mg of P vaginally per day divided in 3 doses, until ET. Progesterone was suspended in the presence of a negative b-hCG test or maintained until 6 to 12 weeks of gestation in the presence of a positive b-hCG test.
Vitrification and Warming Mature oocytes were vitrified within 3 hours after collection and cryo-stored. Embryos were vitrified on day 3 of development. Both vitrification and the warming procedures were performed using the Cryotop method (25). Briefly, vitrification was achieved by exposure of oocytes/embryos initially to the equilibration solution, followed by a 30-second exposure to the vitrification solution. Individual oocytes were then picked up in an extremely small volume (<0.1 mL) of vitrification solution, to facilitate rapid cooling, and placed on top of a very fine polypropylene strip attached to a hard plastic handle. As soon as the oocyte was placed onto the thin polypropylene strip of the Cryotop, it was immediately submerged vertically into liquid nitrogen. Then the thin strip was covered with a hard plastic cover on top of the Cryotop sheet. For warming, the protective cover was removed from the Cryotop while it was still submerged in liquid nitrogen, and the polypropylene strip of the Cryotop was immersed directly into the thawing solution at 37 C for 1 minute. Oocytes or embryos were retrieved and transferred into dilution solution for 3 minutes and then washed twice in the washing solution for 5 minutes each. Tools and solutions required for vitrification and warming processes were obtained from Kitazato. Three hours after warming, intact oocytes were sperm injected, and embryos were cultured until the blastocyst stage. Cleavage-stage embryos were warmed and evaluated; embryos with more than 50% of the cells intact were considered viable. The embryos were incubated until the blastocyst stage when ET was performed.
Embryo Morphology Evaluation Embryo morphology was assessed 16–18 hours after ICSI and on the mornings of days 2, 3, and 5 of embryo development using an inverted Nikon Diaphot microscope (Eclipse TE 300, Nikon) with a Hoffmann modulation contrast system under 400 magnification. To evaluate the cleavage-stage morphology, the following parameters were recorded: number of blastomeres,
percentage of fragmentation, variation in blastomere symmetry, presence of multinucleation, and defects in the zona pellucida and cytoplasm. High-quality cleavage-stage embryos were defined as those with all of the following characteristics: four cells on day 2 or 8–10 cells on day 3, <15% fragmentation, symmetric blastomeres, absence of multinucleation, colorless cytoplasm with moderate granulation and no inclusions, absence of perivitelline space granularity, and absence of zona pellucida dysmorphism. Embryos lacking any of these characteristics were considered to be of low quality. To evaluate blastocyst formation, embryos were given a numerical score from 1 to 6 according to their degree of expansion and hatching status, as follows: 1, an early blastocyst with a blastocoel that was less than half of the volume of the embryo; 2, a blastocyst with a blastocoel that was greater than half of the volume of the embryo; 3, a full blastocyst with a blastocoel that completely filled the embryo; 4, an expanded blastocyst; 5, a hatching blastocyst; and 6, a hatched blastocyst. Full, expanded, hatching, and hatched blastocysts were classified as complete blastocysts.
Statistical Analyses Chi-squared analyses were used to compare embryo quality at the cleavage stage, the chance of blastocyst formation, pregnancy rate, and miscarriage rate among the experimental groups. The fertilization and implantation rates were compared using Student's t tests, and results were expressed as the average SD with the corresponding P values. Logistic and linear regression models were performed to confirm the clinical results. All these analyses were adjusted for endometrial thickness, serum P level on hCG trigger, serum E2 level on hCG trigger, total dose of FSH used for ovarian stimulation, and the quality of transferred embryos (when at least one embryo was transferred it was considered high quality, and when only low-quality embryos were transferred it was considered low quality), because these variables were considered potential confounders in the association between the factors evaluated and clinical ICSI outcomes. Results were expressed as odds ratios, regression coefficients, 95% confidence intervals, and P values. Results were considered to be significant at the 5% critical level (P< .05). The data analysis was performed using the Minitab Statistical Program (version 14).
RESULTS The characteristics of the patients and of the Fresh Oocyte Cycles and Banked Donor Egg Group are described in Table 1. Compared with the Fresh Oocyte Cycles, the ages of men and women and the number of transferred embryos were increased and the fertilization rate was decreased in the Banked Donor Egg Group. Concerning the embryo developmental competence, a decreased quality on days 2 and 3 and a lower blastocyst formation rate was observed among embryos derived from vitrified oocytes (the Banked Donor Egg Group) compared with embryos derived from fresh oocytes (the Fresh Oocyte Cycles Group; Table 1). VOL. - NO. - / - 2016
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TABLE 1 Comparison of the characteristics of patients and cycles, embryo quality on days 2 and 3, and blastocyst formation rate of the Fresh Oocyte Cycles Group and the Banked Donor Egg Group. Group Variable
Fresh oocyte cycles
Banked donor egg
P value
Cycles (n) Embryos (n) Female age (y) Male age (y) No. of follicles Sperm concentration (106/mL) Sperm progressive motility (%) Sperm morphology (%) No. of oocytes No. of metaphase II oocytes Oocyte survival rate (%) Injected oocytes Embryos Fertilization rate Transferred embryos High-quality embryos on D2, % (n) High-quality embryos on D3, % (n) Blastocyst formation, % (n)
425 4,585 31.3 3.3 35.1 5.5 33.7 12.9 46.5 51.0 44.0 15.5 1.5 2.3 25.8 9.7 19.4 7.8 – 12.8 4.8 10.8 4.3 85.4 14.4 1.6 1.0 43.2 (1,984/4,585) 38.6 (1,770/4,585) 41.1 (1,885/4,585)
425 2,128 41.4 5.5 41.2 7.2 – 44.3 39.9 45.4 16.8 1.4 1.8 – – 94.4% 4.3 1.3 3.4 3.0 80.2 18.2 1.8 1.1 31.5 (670/2,128) 30.7 (655/2,128) 36.6 (779/2,128)
< .001 < .001 NC .477 .212 .484 NC NC NC NC NC < .001 < .001 < .001 < .001 < .001
Note: Values are mean SD, unless otherwise noted. D2 ¼ second day of embryo development; D3 ¼ third day of embryo development; NC ¼ not comparable. Almeida Ferreira Braga. Embryo or oocyte vitrification?. Fertil Steril 2016.
Subsequently, the Fresh Oocyte Cycles Group was split into the Thaw Cycles Group and the Fresh Cycles Group. The Thaw Cycles Group and Fresh Cycles Group were then compared with the Banked Donor Egg Group. The embryo survival rate was superior to the oocyte survival rate when the Fresh Cycles Group was compared with the Thaw Cycles Group. A significant increase in the total usable embryos rate was noted for the Fresh Cycles Group when compared with the Banked Donor Egg Group (Table 2). In addition, a significant difference in the clinical outcomes was found: the Thaw Cycles Group had the highest pregnancy and implantation rates, followed by the Banked Donor Egg Group, whereas the Fresh Cycles Group had the lowest rates of pregnancy and implantation (Table 2). These findings were confirmed by binary logistic regression or linear regression analyses, for pregnancy and implantation respectively, adjusted for endometrial thickness, serum P level on hCG trigger, serum E2 level on hCG trigger, total dose of FSH used for ovarian stimulation, and the quality of transferred embryos, using binary logistic regression or linear regression analyses (Table 3).
DISCUSSION The data from the present study show that embryo vitrification leads to better clinical outcomes when compared with outcomes resulting from oocyte vitrification. The data presented here also indicate that cryopreservation cycles using oocytes or embryos result in higher rates of implantation than when fresh embryos are transferred in COS cycles. Although cryopreservation has been established as a useful tool in ART, the slow-freezing method has been shown to
be limited in terms of allowing for successful implantation. As an alternative to slow freezing, the vitrification procedure has been widely applied to human oocytes and embryos, allowing for improvements in cell survival, fertilization, embryo development, and clinical outcomes (26–30). It has been more than two decades since the first successful report of oocyte cryopreservation (31). Because of the length of time since development, and because chromosomal abnormalities, birth defects, and developmental defects are no higher for fresh or vitrified oocytes, this technology is no longer considered experimental by the American Society for Reproductive Medicine and the Society for Assisted Reproductive Technology (32). Although our results have demonstrated that warmed oocytes transferred in endometrial prepared cycles yield better clinical outcomes than fresh embryo transfers in COS cycles, the fertilization rate, embryo quality, and developmental competence was decreased in embryos derived from vitrified oocytes. Moreover, the total usable embryos rate and the pregnancy and implantation rates were lower than that observed when frozen embryos derived from fresh oocytes were transferred. Conversely, previous studies have suggested that oocyte vitrification followed by ICSI is not inferior to fresh insemination with regard to fertilization (33), embryo developmental competence (8, 33), pregnancy rates (8, 26, 33), and live birth (29). In Brazil egg donation may not be conducted for profitable or commercial purposes. Therefore, in Brazil surplus oocytes are only obtained from patients undergoing IVF treatments. Thus, the oocytes available for donation originate from infertile couples. Subsequently, this creates an interesting situation in which, by using an oocyte donor/recipient program, vitrified and nonvitrified oocytes from the same
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ORIGINAL ARTICLE: ASSISTED REPRODUCTION 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649
TABLE 2 Comparison of total usable embryo rate and clinical outcomes considering the status of transferred embryos: [1] fresh oocytes and fresh embryos (Fresh Cycles Group), [2] fresh oocytes and vitrified embryos (Thaw Cycles Group), and [3] vitrified oocytes (Banked Donor Egg Group). Group Variable n Total usable embryo rate, % (n) Pregnancy rate, % (n) Miscarriage rate, % (n) Implantation rate (%)
Fresh cycles
Thaw cycles
Banked donor egg
P value
128 36.4 (501/1,376)a 39.8 (51/128)c 9.4 (5/53) 37.2 41.1f
297 NA 71.4 (212/297)d 10.8 (23/212) 67.3 38.4g
425 39.7 (846/2,128)b 49.6 (211/425)e 12.8 (21/164) 43.0 41.0h
.047 < .001 .679 < .001
Note: Regarding superscript letters, a s b, c s d s e, and f s g s h. NA ¼ not applicable. Almeida Ferreira Braga. Embryo or oocyte vitrification?. Fertil Steril 2016.
cohort can be compared. Another advantage of using samples from a Brazilian donor/recipient program is that the impact of vitrification on the functionality of oocytes derived from infertile couples can be analyzed. This is different from most other published studies, in which data come from oocyte donor populations. The low rates of embryo development observed after oocyte cryopreservation could be explained by possible injury caused by the chilling process. Many organelles, such as meiotic spindles (34), cortical granules (35, 36), and cytoskeleton components (37), are highly sensitive to cryoinjuries. Nevertheless, with the advent of vitrification, which combines ultra-rapid cryopreservation of minimum volumes of sample with a high concentration of cryoprotectants (38), the efficiency and security of many cryopreservation programs has dramatically increased. Another issue to be discussed is the high paternal age of the Recipients Group (Banked Donor Egg Group). High paternal age could explain the low results obtained for this group. Previous studies have suggested that the level of steroid hormones decreases with advanced age (39). A decreased chance of conception within 12 months (40) and an increased risk of miscarriage were also noted (41). However, in a systematic review of the literature, Dain et al. (42) concluded that advanced paternal age is not associated with decreased ART outcomes, including pregnancy rate, miscarriage rate, and live birth rate. Moreover, in the study presented here,
lower seminal parameters were not detected for samples originating from older patients. An interesting point found in this study is that even with lower embryo developmental quality, warmed oocytes transferred in endometrial prepared cycles result in higher pregnancy and implantation rates compared with transfer in fresh COS cycles. This finding strongly suggests that COS impacts endometrial receptivity, which may be a possible cause of implantation failure after ovarian stimulation. Indeed, some studies have suggested that the pregnancy rate is inversely related to serum P levels on the day of hCG administration (43–46). Venetis et al. (47) report that during COS and in the presence of GnRH analogues, elevated P levels might be attributed to an excess number of follicles, with each one producing a normal amount of P consistent with the late follicular phase. Consistent with this, higher serum P levels have been related to greater FSH administration in both GnRH (48) agonist and GnRH antagonist COS protocols (49, 50). It has been demonstrated that elevated P levels on hCG trigger day negatively influence the pregnancy, regardless of the oocyte quality (43). Raised concentrations of P in the late follicular phase are likely to influence the secretory changes of the endometrium, leading to an asynchrony between embryo and endometrial dialogue, which may result in reduced implantation (51).
TABLE 3 Comparison of oocyte/embryo survival rate, total usable embryos rate, and clinical outcomes considering the status of transferred embryos: [1] fresh oocytes and fresh embryos (Fresh Cycles Group), [2] fresh oocytes and vitrified embryos (Thaw Cycles Group), and [3] vitrified oocytes (Banked Donor Egg Group). Group Variable n Oocyte/embryo survival rate, % (n) Total usable embryos rate, % (n) Pregnancy rate, % (n) Miscarriage rate, % (n) Implantation rate, % (n)
Fresh cycles
Thaw cycles
Banked donor egg
P value
128 98.6 (3,165/3,209)a 36.4 (501/1,376)c 39.8 (51/128)e 9.4 (5/53) 37.2 41.1f
297 95.1 (2,128/2,635)b NA 71.4 (212/297)f 10.8 (23/212) 67.3 38.4i
425 NA 39.7 (846/2,128)d 49.6 (211/425)g 12.8 (21/164) 43.0 41.0j
< .001 .047 < .001 .679 < .001
Note: Regarding superscript letters, a s b, c s d, e s f s g, and h s i s j. NA ¼ Not applicable.
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Almeida Ferreira Braga. Embryo or oocyte vitrification?. Fertil Steril 2016.
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On the other hand, patients who respond adequately to COS are more likely to produce better oocytes. Xu et al. (43) reported that the consequences of elevated P levels on IVF cycles outcome in patients with good response are likely to result from a balance between two antagonistic factors: the good embryo quality associated with a good ovarian response, and the impaired receptivity of the endometrium resulting from premature endometrial exposure to P. The oocyte survival rate was slightly lower than the embryo survival rate, possibly because in this study oocytes from infertile patients were used. Even then the oocyte rate was quite satisfactory (95.1%). A therapeutic option for patients with a history of fresh implantation failure is to cryopreserve an entire cohort of embryos and later transfer them into a ‘‘more receptive’’ endometrium (13, 17, 52). Other applications of the freeze-all policy include fertility preservation, reducing the risk of ovarian hyperstimulation syndrome, avoiding the effects of premature P elevation, or awaiting the results of preimplantation genetic screening or preimplantation genetic diagnosis (52, 53). In this investigation the embryo survival rate was extremely high, and the clinical outcomes after embryo vitrification and warming are encouraging. In conclusion, our results indicate that oocyte vitrification followed by ICSI leads to lower embryo developmental competence when compared with fresh insemination procedures. However, pregnancy and implantation rates are higher when embryos are transferred into a ‘‘more receptive’’ endometrium free of the adverse effects of gonadotropin. Therefore, the use of this technology for egg donation and cryobanking programs is a good alternative. In addition, our findings demonstrate that the embryo freeze-all policy leads to exceptional clinical outcomes and is an excellent alternative to fresh embryo transfer.
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ORIGINAL ARTICLE: ASSISTED REPRODUCTION 827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885
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Freeze-all, oocyte vitrification, or fresh embryo transfer? Lessons from an egg-sharing donation program
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synchronization between the donor cycle and the recipient endometrium (8). In addition, the use of donor oocyte cryobanking may provide patients with more choices when selecting oocytes, more flexibility in timing pregnancy, and potential reductions in the cost of treatments (1, 9). It has been reported that controlled ovarian stimulation (COS) is associated with impaired endometrial receptivity in IVF cycles (10–13). This may be explained by an embryo– endometrium asynchrony: there have been reports of a positive correlation between embryo developmental pace and implantation in fresh autologous IVF cycles. This asynchrony has not, however, been detected in oocyte donation cycles or cycles using frozen–thawed embryos (14, 15). In frozen–thawed cycles, when COS is not performed, pregnancy rates have been reported to be higher than in fresh autologous cycles (16). Considering the adverse effects of COS on endometrial receptivity, the freeze-all policy has emerged as an alternative to fresh embryo transfer to improve IVF outcomes (17, 18). With the freeze-all policy, an entire cohort of embryos is cryopreserved to be transferred later during a natural cycle, or during a cycle with hormonal replacement for endometrial priming. This provides a more physiologic environment for ET (18). It has previously been suggested that oocyte cryopreservation could result in disruption of the oolema molecular machinery required for normal segregation of chromosomes (19, 20). However, other studies have suggested that embryo quality is not affected by the oocyte vitrification procedure. Similar to embryos derived from fresh oocytes, it has been shown that vitrified oocytes preserve the potential to be fertilized and to develop into high-quality blastocysts (21). Moreover, in a paired randomized, controlled trial, Forman et al (22) have demonstrated that oocyte vitrification does not increase the rate of aneuploidy or diminish the implantation potential of viable blastocysts. Likewise, embryos can be consistently cryopreserved and warmed to yield pregnancy outcomes similar to those achieved after the use of fresh embryos (23). To date, however, little is known about the optimal oocyte/embryo stage that should be used for vitrification. Although it has been strongly suggested that ET during a natural or endometrium-prepared cycle is a better approach than transfer during a COS cycle, the question about the vitrification of oocytes or embryos remains under debate. Therefore, the goal of the present study was to compare the outcomes of ETs using cryopreserved embryos or cryopreserved oocytes in endometrial primed cycles by making use of well-controlled oocyte sampling (i.e., derived from oocyte donor patients). A secondary objective was to compare the quality of embryos derived from fresh or vitrified oocytes. Last, this study aimed to confirm the superiority of the freezeall protocol over fresh embryo transfer during COS cycles.
MATERIALS AND METHODS Study Design This study was performed in a privately assisted fertilization center in Brazil and included 8,210 viable mature oocytes obtained from 425 oocyte donors: 5,440 were used for the
donors’ own cycles (Fresh Oocyte Cycles Group) and 2,770 were cryobanked for 425 oocyte recipients (Banked Donor Egg Group). After warming, 2,635 oocytes survived. All oocytes were submitted to sperm injection, resulting in 4,585 embryos obtained from the Fresh Oocyte Cycles Group and 2,128 embryos obtained from the Banked Donor Egg Group. Concerning the Fresh Oocyte Cycles Group, the embryos were either cryopreserved and transferred in a subsequent cycle (Thaw Cycles Group, n ¼ 297 cycles, 3,209 embryos) or transferred during a fresh cycle (Fresh Cycles Group, n ¼ 128 cycles, 1,307 embryos). Therefore the Fresh Oocyte Cycles Group was split into two other groups: the Thaw Cycles Group and the Fresh Cycles Group (Fig. 1A). For the recipient cycles, embryos derived from vitrified oocytes were transferred (Banked Donor Egg Group, n ¼ 425 cycles, 2,128 embryos; Fig. 1A). All of the embryos were evaluated 16–18 hours after intracytoplasmic sperm injection (ICSI) on days 2, 3, and 5 of development when ET was performed. To evaluate the efficiency of the use of fresh or vitrified oocytes, the groups Fresh Oocyte Cycles and Banked Donor Egg Group were compared for fertilization rate, embryo quality on days 2 and 3, and blastocyst formation rate (Fig. 1B). The oocyte/embryo survival rate, total usable embryos rate, implantation rate, pregnancy rate, and miscarriage rate were compared among the Thaw Cycles Group, Fresh Cycles Group, and Banked Donor Egg Group (Fig. 1B). The oocyte/embryo survival rate was calculated by the number of surviving oocytes or embryos after warming divided by the number of vitrified oocytes or embryos. Total usable embryos was calculated by the number of transferred embryo plus the number of supernumerary cryopreserved embryos for future use dived by the total number of obtained embryos. The implantation rate was defined as the total number of gestational sacs divided by the total number of embryos that were transferred. Clinical pregnancy was defined as the presence of a gestational sac that could be visualized using ultrasound 4–6 weeks after ET, and miscarriage was defined as pregnancy with a total loss of gestational sacs before 20 weeks’ gestation. All of the cases with severe spermatogenic alterations, including frozen and surgically retrieved sperm, were excluded from the study. Written informed consent, in which patients agreed to share the outcomes of their cycles for research purposes, was obtained; the study was approved by the local institutional review board.
Controlled Ovarian Stimulation and Laboratory Procedures Controlled ovarian stimulation was achieved by pituitary blockage using a GnRH antagonist (Cetrotide, Serono); ovarian stimulation was performed using recombinant FSH (Gonal-F, Serono). Follicular growth was monitored using transvaginal ultrasound examination starting on day 4 of gonadotropin administration. When adequate follicular growth and serum E2 levels were observed, recombinant hCG VOL. - NO. - / - 2016
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Fertility and Sterility® 237 FIGURE 1 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 Q5 Description of experimental groups and comparisons. 285 Almeida Ferreira Braga. Embryo or oocyte vitrification? Fertil Steril 2016. 286 287 288 289 (Ovidrel, Serono) was administered to trigger the final 290 follicular maturation. The oocytes were collected 35 hours 291 after hCG administration through transvaginal ultrasound 292 ovum pick-up. 293 The recovered oocytes were assessed to determine their 294 nuclear status, and those in metaphase II were submitted to 295 ICSI following routine procedures (24).
A
B
Endometrial Preparation After menses, endometrial development was followed by ultrasound examination, and the patients receive 200 mg of transdermal 17b-E2 every 3 days (Estradot, Noven Pharmaceuticals). Approximately 14 days after initiation of E2 administration, serum E2 levels and endometrial thickness
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were determined. When endometrium showed proliferative morphology and thickness of at least 7.5 mm, 600 mg of P was vaginally administered per day. The administration of P was divided into 3 doses that were started (Utrogestan, Farmoquímica) and maintained until ET. Both 17b-E2 and P were administered concomitantly for 10 days after ET. The 17b-E2 and P treatments were suspended in the presence of a negative b-hCG test. In the presence of a positive bhCG test, the 17b-E2 and P treatments were maintained until weeks 6 and 12 of gestation, respectively. For COS cycles and fresh embryo transfers, on the day of ovum pick-up, patients received 600 mg of P vaginally per day divided in 3 doses, until ET. Progesterone was suspended in the presence of a negative b-hCG test or maintained until 6 to 12 weeks of gestation in the presence of a positive b-hCG test.
Vitrification and Warming Mature oocytes were vitrified within 3 hours after collection and cryo-stored. Embryos were vitrified on day 3 of development. Both vitrification and the warming procedures were performed using the Cryotop method (25). Briefly, vitrification was achieved by exposure of oocytes/embryos initially to the equilibration solution, followed by a 30-second exposure to the vitrification solution. Individual oocytes were then picked up in an extremely small volume (<0.1 mL) of vitrification solution, to facilitate rapid cooling, and placed on top of a very fine polypropylene strip attached to a hard plastic handle. As soon as the oocyte was placed onto the thin polypropylene strip of the Cryotop, it was immediately submerged vertically into liquid nitrogen. Then the thin strip was covered with a hard plastic cover on top of the Cryotop sheet. For warming, the protective cover was removed from the Cryotop while it was still submerged in liquid nitrogen, and the polypropylene strip of the Cryotop was immersed directly into the thawing solution at 37 C for 1 minute. Oocytes or embryos were retrieved and transferred into dilution solution for 3 minutes and then washed twice in the washing solution for 5 minutes each. Tools and solutions required for vitrification and warming processes were obtained from Kitazato. Three hours after warming, intact oocytes were sperm injected, and embryos were cultured until the blastocyst stage. Cleavage-stage embryos were warmed and evaluated; embryos with more than 50% of the cells intact were considered viable. The embryos were incubated until the blastocyst stage when ET was performed.
Embryo Morphology Evaluation Embryo morphology was assessed 16–18 hours after ICSI and on the mornings of days 2, 3, and 5 of embryo development using an inverted Nikon Diaphot microscope (Eclipse TE 300, Nikon) with a Hoffmann modulation contrast system under 400 magnification. To evaluate the cleavage-stage morphology, the following parameters were recorded: number of blastomeres,
percentage of fragmentation, variation in blastomere symmetry, presence of multinucleation, and defects in the zona pellucida and cytoplasm. High-quality cleavage-stage embryos were defined as those with all of the following characteristics: four cells on day 2 or 8–10 cells on day 3, <15% fragmentation, symmetric blastomeres, absence of multinucleation, colorless cytoplasm with moderate granulation and no inclusions, absence of perivitelline space granularity, and absence of zona pellucida dysmorphism. Embryos lacking any of these characteristics were considered to be of low quality. To evaluate blastocyst formation, embryos were given a numerical score from 1 to 6 according to their degree of expansion and hatching status, as follows: 1, an early blastocyst with a blastocoel that was less than half of the volume of the embryo; 2, a blastocyst with a blastocoel that was greater than half of the volume of the embryo; 3, a full blastocyst with a blastocoel that completely filled the embryo; 4, an expanded blastocyst; 5, a hatching blastocyst; and 6, a hatched blastocyst. Full, expanded, hatching, and hatched blastocysts were classified as complete blastocysts.
Statistical Analyses Chi-squared analyses were used to compare embryo quality at the cleavage stage, the chance of blastocyst formation, pregnancy rate, and miscarriage rate among the experimental groups. The fertilization and implantation rates were compared using Student's t tests, and results were expressed as the average SD with the corresponding P values. Logistic and linear regression models were performed to confirm the clinical results. All these analyses were adjusted for endometrial thickness, serum P level on hCG trigger, serum E2 level on hCG trigger, total dose of FSH used for ovarian stimulation, and the quality of transferred embryos (when at least one embryo was transferred it was considered high quality, and when only low-quality embryos were transferred it was considered low quality), because these variables were considered potential confounders in the association between the factors evaluated and clinical ICSI outcomes. Results were expressed as odds ratios, regression coefficients, 95% confidence intervals, and P values. Results were considered to be significant at the 5% critical level (P< .05). The data analysis was performed using the Minitab Statistical Program (version 14).
RESULTS The characteristics of the patients and of the Fresh Oocyte Cycles and Banked Donor Egg Group are described in Table 1. Compared with the Fresh Oocyte Cycles, the ages of men and women and the number of transferred embryos were increased and the fertilization rate was decreased in the Banked Donor Egg Group. Concerning the embryo developmental competence, a decreased quality on days 2 and 3 and a lower blastocyst formation rate was observed among embryos derived from vitrified oocytes (the Banked Donor Egg Group) compared with embryos derived from fresh oocytes (the Fresh Oocyte Cycles Group; Table 1). VOL. - NO. - / - 2016
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TABLE 1 Comparison of the characteristics of patients and cycles, embryo quality on days 2 and 3, and blastocyst formation rate of the Fresh Oocyte Cycles Group and the Banked Donor Egg Group. Group Variable
Fresh oocyte cycles
Banked donor egg
P value
Cycles (n) Embryos (n) Female age (y) Male age (y) No. of follicles Sperm concentration (106/mL) Sperm progressive motility (%) Sperm morphology (%) No. of oocytes No. of metaphase II oocytes Oocyte survival rate (%) Injected oocytes Embryos Fertilization rate Transferred embryos High-quality embryos on D2, % (n) High-quality embryos on D3, % (n) Blastocyst formation, % (n)
425 4,585 31.3 3.3 35.1 5.5 33.7 12.9 46.5 51.0 44.0 15.5 1.5 2.3 25.8 9.7 19.4 7.8 – 12.8 4.8 10.8 4.3 85.4 14.4 1.6 1.0 43.2 (1,984/4,585) 38.6 (1,770/4,585) 41.1 (1,885/4,585)
425 2,128 41.4 5.5 41.2 7.2 – 44.3 39.9 45.4 16.8 1.4 1.8 – – 94.4% 4.3 1.3 3.4 3.0 80.2 18.2 1.8 1.1 31.5 (670/2,128) 30.7 (655/2,128) 36.6 (779/2,128)
< .001 < .001 NC .477 .212 .484 NC NC NC NC NC < .001 < .001 < .001 < .001 < .001
Note: Values are mean SD, unless otherwise noted. D2 ¼ second day of embryo development; D3 ¼ third day of embryo development; NC ¼ not comparable. Almeida Ferreira Braga. Embryo or oocyte vitrification?. Fertil Steril 2016.
Subsequently, the Fresh Oocyte Cycles Group was split into the Thaw Cycles Group and the Fresh Cycles Group. The Thaw Cycles Group and Fresh Cycles Group were then compared with the Banked Donor Egg Group. The embryo survival rate was superior to the oocyte survival rate when the Fresh Cycles Group was compared with the Thaw Cycles Group. A significant increase in the total usable embryos rate was noted for the Fresh Cycles Group when compared with the Banked Donor Egg Group (Table 2). In addition, a significant difference in the clinical outcomes was found: the Thaw Cycles Group had the highest pregnancy and implantation rates, followed by the Banked Donor Egg Group, whereas the Fresh Cycles Group had the lowest rates of pregnancy and implantation (Table 2). These findings were confirmed by binary logistic regression or linear regression analyses, for pregnancy and implantation respectively, adjusted for endometrial thickness, serum P level on hCG trigger, serum E2 level on hCG trigger, total dose of FSH used for ovarian stimulation, and the quality of transferred embryos, using binary logistic regression or linear regression analyses (Table 3).
DISCUSSION The data from the present study show that embryo vitrification leads to better clinical outcomes when compared with outcomes resulting from oocyte vitrification. The data presented here also indicate that cryopreservation cycles using oocytes or embryos result in higher rates of implantation than when fresh embryos are transferred in COS cycles. Although cryopreservation has been established as a useful tool in ART, the slow-freezing method has been shown to
be limited in terms of allowing for successful implantation. As an alternative to slow freezing, the vitrification procedure has been widely applied to human oocytes and embryos, allowing for improvements in cell survival, fertilization, embryo development, and clinical outcomes (26–30). It has been more than two decades since the first successful report of oocyte cryopreservation (31). Because of the length of time since development, and because chromosomal abnormalities, birth defects, and developmental defects are no higher for fresh or vitrified oocytes, this technology is no longer considered experimental by the American Society for Reproductive Medicine and the Society for Assisted Reproductive Technology (32). Although our results have demonstrated that warmed oocytes transferred in endometrial prepared cycles yield better clinical outcomes than fresh embryo transfers in COS cycles, the fertilization rate, embryo quality, and developmental competence was decreased in embryos derived from vitrified oocytes. Moreover, the total usable embryos rate and the pregnancy and implantation rates were lower than that observed when frozen embryos derived from fresh oocytes were transferred. Conversely, previous studies have suggested that oocyte vitrification followed by ICSI is not inferior to fresh insemination with regard to fertilization (33), embryo developmental competence (8, 33), pregnancy rates (8, 26, 33), and live birth (29). In Brazil egg donation may not be conducted for profitable or commercial purposes. Therefore, in Brazil surplus oocytes are only obtained from patients undergoing IVF treatments. Thus, the oocytes available for donation originate from infertile couples. Subsequently, this creates an interesting situation in which, by using an oocyte donor/recipient program, vitrified and nonvitrified oocytes from the same
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532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590
ORIGINAL ARTICLE: ASSISTED REPRODUCTION 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649
TABLE 2 Comparison of total usable embryo rate and clinical outcomes considering the status of transferred embryos: [1] fresh oocytes and fresh embryos (Fresh Cycles Group), [2] fresh oocytes and vitrified embryos (Thaw Cycles Group), and [3] vitrified oocytes (Banked Donor Egg Group). Group Variable n Total usable embryo rate, % (n) Pregnancy rate, % (n) Miscarriage rate, % (n) Implantation rate (%)
Fresh cycles
Thaw cycles
Banked donor egg
P value
128 36.4 (501/1,376)a 39.8 (51/128)c 9.4 (5/53) 37.2 41.1f
297 NA 71.4 (212/297)d 10.8 (23/212) 67.3 38.4g
425 39.7 (846/2,128)b 49.6 (211/425)e 12.8 (21/164) 43.0 41.0h
.047 < .001 .679 < .001
Note: Regarding superscript letters, a s b, c s d s e, and f s g s h. NA ¼ not applicable. Almeida Ferreira Braga. Embryo or oocyte vitrification?. Fertil Steril 2016.
cohort can be compared. Another advantage of using samples from a Brazilian donor/recipient program is that the impact of vitrification on the functionality of oocytes derived from infertile couples can be analyzed. This is different from most other published studies, in which data come from oocyte donor populations. The low rates of embryo development observed after oocyte cryopreservation could be explained by possible injury caused by the chilling process. Many organelles, such as meiotic spindles (34), cortical granules (35, 36), and cytoskeleton components (37), are highly sensitive to cryoinjuries. Nevertheless, with the advent of vitrification, which combines ultra-rapid cryopreservation of minimum volumes of sample with a high concentration of cryoprotectants (38), the efficiency and security of many cryopreservation programs has dramatically increased. Another issue to be discussed is the high paternal age of the Recipients Group (Banked Donor Egg Group). High paternal age could explain the low results obtained for this group. Previous studies have suggested that the level of steroid hormones decreases with advanced age (39). A decreased chance of conception within 12 months (40) and an increased risk of miscarriage were also noted (41). However, in a systematic review of the literature, Dain et al. (42) concluded that advanced paternal age is not associated with decreased ART outcomes, including pregnancy rate, miscarriage rate, and live birth rate. Moreover, in the study presented here,
lower seminal parameters were not detected for samples originating from older patients. An interesting point found in this study is that even with lower embryo developmental quality, warmed oocytes transferred in endometrial prepared cycles result in higher pregnancy and implantation rates compared with transfer in fresh COS cycles. This finding strongly suggests that COS impacts endometrial receptivity, which may be a possible cause of implantation failure after ovarian stimulation. Indeed, some studies have suggested that the pregnancy rate is inversely related to serum P levels on the day of hCG administration (43–46). Venetis et al. (47) report that during COS and in the presence of GnRH analogues, elevated P levels might be attributed to an excess number of follicles, with each one producing a normal amount of P consistent with the late follicular phase. Consistent with this, higher serum P levels have been related to greater FSH administration in both GnRH (48) agonist and GnRH antagonist COS protocols (49, 50). It has been demonstrated that elevated P levels on hCG trigger day negatively influence the pregnancy, regardless of the oocyte quality (43). Raised concentrations of P in the late follicular phase are likely to influence the secretory changes of the endometrium, leading to an asynchrony between embryo and endometrial dialogue, which may result in reduced implantation (51).
TABLE 3 Comparison of oocyte/embryo survival rate, total usable embryos rate, and clinical outcomes considering the status of transferred embryos: [1] fresh oocytes and fresh embryos (Fresh Cycles Group), [2] fresh oocytes and vitrified embryos (Thaw Cycles Group), and [3] vitrified oocytes (Banked Donor Egg Group). Group Variable n Oocyte/embryo survival rate, % (n) Total usable embryos rate, % (n) Pregnancy rate, % (n) Miscarriage rate, % (n) Implantation rate, % (n)
Fresh cycles
Thaw cycles
Banked donor egg
P value
128 98.6 (3,165/3,209)a 36.4 (501/1,376)c 39.8 (51/128)e 9.4 (5/53) 37.2 41.1f
297 95.1 (2,128/2,635)b NA 71.4 (212/297)f 10.8 (23/212) 67.3 38.4i
425 NA 39.7 (846/2,128)d 49.6 (211/425)g 12.8 (21/164) 43.0 41.0j
< .001 .047 < .001 .679 < .001
Note: Regarding superscript letters, a s b, c s d, e s f s g, and h s i s j. NA ¼ Not applicable.
Q3
Almeida Ferreira Braga. Embryo or oocyte vitrification?. Fertil Steril 2016.
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650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708
Fertility and Sterility® 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767
On the other hand, patients who respond adequately to COS are more likely to produce better oocytes. Xu et al. (43) reported that the consequences of elevated P levels on IVF cycles outcome in patients with good response are likely to result from a balance between two antagonistic factors: the good embryo quality associated with a good ovarian response, and the impaired receptivity of the endometrium resulting from premature endometrial exposure to P. The oocyte survival rate was slightly lower than the embryo survival rate, possibly because in this study oocytes from infertile patients were used. Even then the oocyte rate was quite satisfactory (95.1%). A therapeutic option for patients with a history of fresh implantation failure is to cryopreserve an entire cohort of embryos and later transfer them into a ‘‘more receptive’’ endometrium (13, 17, 52). Other applications of the freeze-all policy include fertility preservation, reducing the risk of ovarian hyperstimulation syndrome, avoiding the effects of premature P elevation, or awaiting the results of preimplantation genetic screening or preimplantation genetic diagnosis (52, 53). In this investigation the embryo survival rate was extremely high, and the clinical outcomes after embryo vitrification and warming are encouraging. In conclusion, our results indicate that oocyte vitrification followed by ICSI leads to lower embryo developmental competence when compared with fresh insemination procedures. However, pregnancy and implantation rates are higher when embryos are transferred into a ‘‘more receptive’’ endometrium free of the adverse effects of gonadotropin. Therefore, the use of this technology for egg donation and cryobanking programs is a good alternative. In addition, our findings demonstrate that the embryo freeze-all policy leads to exceptional clinical outcomes and is an excellent alternative to fresh embryo transfer.
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