ORIGINAL ARTICLE: ASSISTED REPRODUCTION

Human oocyte cryopreservation with slow freezing versus vitrification. Results from the National Italian Registry data, 2007–2011 Paolo Emanuele Levi Setti, M.D.,a Eleonora Porcu, M.D.,b Pasquale Patrizio, M.D, M.B.E.,c Vincenzo Vigiliano, B.Sc.,d Roberto de Luca, B.Sc.,d Paola d’Aloja, Ph.D.,d Roberta Spoletini, B.Sc.,d and Giulia Scaravelli, M.D., Ph.D.d a

Humanitas Fertility Center, Division of Gynaecology and Reproductive Medicine, Department of Gynaecology, Humanitas Research Hospital, Rozzano, Italy; b Infertility and IVF Center, S. Orsola–Malpighi University Hospital, University of Bologna, Bologna, Italy; c Department of Obstetrics, Gynecology, and Reproductive Sciences, School of Medicine, Yale University, New Haven, Connecticut; and d ART Italian National Registry, National Center for Epidemiology, Surveillance, and Health Promotion, National Health Institute, Rome, Italy

Objective: To compare mature human oocytes cryopreservation with slow freezing (SF) and vitrification (VT) in infertile couples. Design: Retrospective study of national Italian data submitted during the period 2007–2011. Setting: National ART registry. Patient(s): Infertile patients with supernumerary oocytes. Intervention(s): Thawing or warming of cryopreserved oocytes and ICSI. Main Outcome Measure(s): oocyte survival, fertilization, implantation and clinical pregnancy rate between SF and VT. Result(s): A total of 14,328 cycles with 11,599 transfers, 1,850 pregnancies, 1,168 deliveries and 1,342 babies born were analyzed from 146 reporting centers (range of cycles 1–1,255 per center). The SF oocytes' survival rate was lower than in VT (51.1% vs. 63.1%). Fertilization rate was significantly higher in SF than in VT (SF 71.6% vs. VT 70.1%). VT showed a significantly higher pregnancy rate, both per started cycle (14.4% vs. 12.0%) and per transfer (18.0% vs. 14.8%), and implantation rate (9.5% vs. 8.1%) than SF. However, the range and median pregnancy rate per started cycle were, respectively, 0%–50% and 7.7% in SF and 0%–100% and 6.7% in VT. Conclusion(s): VT showed a statistically significant higher performance than SF. As with other ART procedures, the results are not homogeneous among clinics and protocols, but the confirm Use your smartphone the clinical value of oocyte cryopreservation in infertile patients. (Fertil SterilÒ 2014;-:-–-. to scan this QR code Ó2014 by American Society for Reproductive Medicine.) and connect to the Key Words: Oocyte cryopreservation, methods, ART safety, pregnancy outcome Discuss: You can discuss this article with its authors and other ASRM members at http:// fertstertforum.com/settip-human-oocyte-cryopreservation-national-italian-registry/

O

ocyte cryopreservation is an important option for individuals planning to undergo IVF treatment, and newly developed protocols have greatly improved the application of this procedure (1–9). Two

different methods of oocyte cryopreservation are currently used: the long-established technique termed slow freezing (SF), and the more recent process called vitrification (VT). With the SF protocol the oocytes are gradu-

Received February 15, 2014; revised March 26, 2014; accepted March 27, 2014. P.E.L.S. has nothing to disclose. E.P. has nothing to disclose. P.P. has nothing to disclose. V.V. has nothing to disclose. R.d.L. has nothing to disclose. P.d’A. has nothing to disclose. R.S. has nothing to disclose. G.S. has nothing to disclose. Reprint requests: Paolo Emanuele Levi Setti, M.D., Humanitas Fertility Center, Division of Gynaecology and Reproductive Medicine, Department of Gynaecology, Humanitas Research Hospital, 20084 Rozzano (Milano), Italy (E-mail: [email protected]). Fertility and Sterility® Vol. -, No. -, - 2014 0015-0282/$36.00 Copyright ©2014 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2014.03.052 VOL. - NO. - / - 2014

discussion forum for this article now.*

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ally frozen in the presence of low concentrations of cryoprotectant agents, which minimize the risk of intracellular ice formation and structural damage. The VT method uses a high initial concentration of cryoprotectants and ultrarapid cooling to transform the cell into a glass-like state to avoid the formation of ice crystals (10, 11). The VT procedure seems to be superior to SF and similar to the use of fresh oocytes (12, 13). In Italy, the use of oocyte cryopreservation increased rapidly after legal restrictions (14–17) limited the number of oocytes to be 1

ORIGINAL ARTICLE: ASSISTED REPRODUCTION inseminated per cycle to a maximum of three, and banned embryo cryopreservation. Since 2005, the Italian Registry of Assisted Reproduction Technologies at the Italian National Health Institute, has collected data on ART cycles, including those performed with cryopreserved oocytes (18). From 2007 the Registry also collected data on different oocyte cryopreservation methods (SF vs. VT). To compare the efficiency of slow freezing and vitrification, we analyzed IVF cycles using cryopreserved oocytes from 2007 to 2011. Importantly, in May 2009 the Italian Constitutional Court acting on defending the best interest of women undergoing IVF treatments modified some of the legal restrictions on IVF, leaving to the clinician the choice of the most appropriate IVF treatment. Since this modification, the decision on number of oocytes to inseminate and embryos to transfer has been assigned to the reproductive specialist (14, 19). This detail becomes important when judging differences in clinical outcomes of cryopreserved oocytes before and after the decision of the Constitutional Court, because before the change, more oocytes per patient were available for cryopreservation and fewer were usable in fresh cycles. After the law was modified, more oocytes per patient were inseminated in fresh cycles and fewer were made available for cryopreservation. This could have skewed the interpretation of the results for oocyte cryopreservation between prior and after the Law was modified. In the present study, we performed a retrospective analysis of data on clinical outcomes of 14,328 ART procedures using cryopreserved oocytes (8,927 SF cycles, 5,401 VT cycles). All Italian ART clinics that performed cycles on cryopreserved oocytes (CO) and submitted data to the National Registry from 2007 to 2011 contributed to this study. Post-thawing/warming survival rate, number of transferable embryos per cycle, implantation rates per cycle, pregnancy rates per cycle and per transfer, and live births per cycle and transfer were evaluated as primary outcomes. Secondary outcomes analyzed were pregnancy rates per age groups, negative pregnancy outcomes per monitored pregnancies, and numbers of warmed/thawed oocytes needed to have a live birth. To investigate differences among centers, median and range of pregnancy rates were also analyzed.

MATERIALS AND METHODS Aggregate data of IVF cycles performed on cryopreserved oocytes from 2007 to 2011 were analyzed. Data were collected with the use of an internet web site (www.iss.it/rpma) a resource set up by the ART National Registry at the National Center for Epidemiology, Surveillance, and Health Promotion of the Istituto Superiore di Sanita in Roma, Italy. Records were stored on a secure server and password protected. This work did not require Institutional Review Board approval, because it consisted of analysis of data from the Italian National ART repository not linked to patient identifiers. In accordance with Law 40/2004, no more than three embryos were generated after oocyte thawing/warming from 2007 to May 2009. After the latter date the restriction on the maximum of three oocytes to be fertilized was lifted. Implantation rates were calculated by dividing the number of gestational sacs by the number of transferred embryos (20). Ectopic pregnancies were included. A live birth was defined as a viable infant born at R24 weeks of gestation.

Statistical Analysis Data were analyzed with the use of SPSS Statistics 17.0. Percentages of transferred embryos per inseminated oocytes, implantation rate, and pregnancy rate per started cycle and per transfer, delivery rate, spontaneous or therapeutic abortions, ectopic pregnancies per monitored pregnancy, and malformed babies per live birth were calculated. These parameters were compared between thawed and warmed oocyte cycles with the use of crude odds ratio (OR) of exposure to SF/VT protocol and 95% confidence interval (CI). P< .05 was considered to be statistically significant.

RESULTS During the 5-year study period, the Italian ART centers performed a total of 14,328 cycles using CO, of which 62.3% (8,927) used SF/thawing and 37.7% (5,401) VT/warming. The number of ART clinics that carried out CO cycles differed from year to year (Table 1), increasing from 93 in 2007 to 120 in 2011. In more detail, the number of CO cycles adopting VT procedure increased from 568 in 2007 to 1,644 in 2011. Conversely, the number of cycles using SF decreased from

TABLE 1 Numbers of clinics and cycles performed with slow freezing, vitrification, and fresh cycles and number of cycles performed by each Center (min– max). Slow freezing Year 2007 2008 2009 2010 2011a Total

Vitrification

Total cycles

Fresh cycles

Centers

Cycles

Range

Centers

Cycles

Range

Centers

Cycles

Range

Centers

Cycles

Range

85 88 88 75 64 109

2,426 2,625 1,916 1,097 863 8,927

1–270 1–304 1–165 1–81 1–80 1–805

30 41 60 69 88 102

568 659 1,186 1,344 1,644 5,401

1–252 1–225 1–162 1–143 1–145 1–718

93 104 114 109 120 146

2,994 3,284 3,102 2,441 2,507 14,328

1–270 1–304 1–325 1–191 1–177 1–1,255

181 185 180 174 179 204

40,005 44,037 47,911 52,661 56,086 240,700

2–1,415 5–1,599 7–1,702 7–1,847 2–1,897 2–8,460

a

In 2011 a minimum of one cycle of oocyte thawing/warming was performed by 120/179 (67%) of the reporting centers, showing a growth in the number of centers offering oocyte cryopreservation procedures. Levi Setti. OC by slow freezing or vitrification. Fertil Steril 2014.

2

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< .001 .162 .061 .481 .078 .037 1.44 (1.25–1.65) 0.92 (0.82–1.03) 1.09 (1.00–1.20) 1.03 (0.94–1.13) 0.92 (0.84–1.01) 0.96 (0.92–1.00) 76.6 68.6 71.9 66.6 64.4 68.0 1,049/1,370 1,155/1,683 2,507/3,488 2,951/4,430 3,695/5,742 11,357/16,713 4,173/6,008 4,536/6,445 3,868/5,523 2,393/3,633 1,904/2,873 16,874/24,482

VOL. - NO. - / - 2014

Levi Setti. OC by slow freezing or vitrification. Fertil Steril 2014.

Note: CI ¼ confidence interval; OR ¼ odds ratio; SF ¼ slow freezing; VT ¼ vitrification.

2007 2008 2009 2010 2011 Overall

4,173/12,573 4,536/13,592 3,868/10,821 2,393/6,068 1,904/4,860 16,874/47,914

33.2 33.4 35.7 39.4 39.2 35.2

1,049/2,317 1,155/2,949 2,507/5,707 2,951/6,906 3,695/8,625 11,357/26,504

45.3 39.2 43.9 42.7 42.8 42.9

1.67 (1.52–1.82) 1.29 (1.18–1.40) 1.41 (1.32–1.50) 1.15 (1.07–1.23) 1.16 (1.08–1.25) 1.38 (1.34–1.42)

< .001 < .001 < .001 < .001 < .001 < .001

69.5 70.4 70.0 65.9 66.3 68.9

OR (95% CI), SF (SF [ 1) vs. VT % VT SF P value OR (95% CI), SF (SF [ 1) vs. VT % VT % SF Year

Total embryos per thawed/warmed oocytes

Total embryos per thawed/warmed oocytes and per injected oocytes following slow freezing and vitrification cycles.

TABLE 2

%

Total embryos/injected oocytes

P value

Fertility and Sterility® 2,426 in 2007 to 863 in 2011. The overall number of CO cycles, however, was lower after Law 40 was amended to allow using higher numbers of oocyte for insemination in fresh cycles. In 2011 at least one thawing/warming cycle was performed by 120/179 (67%) of reporting centers (Table 1). The number of CO procedures carried out during the period (2007–2011) ranged from 1 to 1,255 per reporting clinic, 1–805 with SF and 1–718 with VT (Table 1). A total of 74,418 cryopreserved oocytes were thawed/warmed, 64.4% of which (47,914) were in the SF group and 35.6% (26,504) in the VT group. Overall, fewer oocytes survived the SF/ thawing process than VT/warming (51.1% vs. 63.1%, OR 1.63; P< .001). This percentage, however, does not represent the crude survival rate after thawing/warming of the oocytes, because in the period from 2007 to May 2009 only three oocytes could be inseminated, even if more oocytes survived the thawing procedure. The VT survival rate was significantly higher in all of the years considered. To evaluate which step can be more critical for the efficiency of the entire process, the number of embryos obtained per thawed/warmed oocyte and per inseminated oocyte was analyzed. Table 2 summarizes that ‘‘obtained embryos’’ per thawed (SF) and warmed (VT) oocyte were 35.2% and 42.9% (OR 1.38), respectively, whereas ‘‘obtained embryos’’ per inseminated oocyte were 68.9% and 68% (OR 0.96), respectively. These cumulative data confirm that VT survival rate is significantly better than SF, but the fertilization rate is not different (Table 2). Implantation rates (Table 3) were higher with embryos from VT/warmed oocytes in each year, but significantly higher only in 2009. The overall results (Table 3) show a 1.4% significantly higher VT implantation rate with an OR of 1.20 (95% CI 1.09–1.31). A total of 1,850 pregnancies were obtained (Table 4), 1,074 from SF and 776 from VT. The clinical pregnancy rates per cycle and per transfer (Table 4) significantly increased during the study period, from 10.5% to 13.8% per cycle and from 13.1% to 16.7% per transfer with SF, and from 12.9% to 14.2% per cycle and 15.1 to 17.9% per transfer with VT. The pregnancy rates for cycle and transfer were significantly higher with VT in 2009, but not in 2007, 2008, 2010, and 2011. The overall pregnancy rate per cycle was, however, 2.2% significantly higher in VT (OR 1.23, CI 1.11–1.35) and per transfer 3.2% (OR 1.26, CI 1.14–1.40). To analyze the differences between centers performing SF and VT, the number of SF and VT cycles performed by each center and the median and range of pregnancy rates per cycle and transfer were calculated in each reported year and in the study period (Table 4). The results showed large differences among centers, with some ART programs performing only 1 cycle in the 5-year period and others performing up to 1,255 cycles of both investigated procedures. The overall mean pregnancy rates per cycle and transfer were 12.0% and 14.8% for SF and 14.4% and 18.0% for VT. The range and median values of the pregnancy rate per cycle were 0%–50% and 7.7% in SF and 0%–100% and 6.7% in VT. The overall percentage of multiple pregnancies was 14.9% (275/1,850), with no significant differences between SF (15.7%, 169/1,074) and VT (13.7%, 106/776). 3

ORIGINAL ARTICLE: ASSISTED REPRODUCTION

TABLE 3 Implantation rates following slow freezing, vitrification, and fresh cycles. Year 2007 2008 2009 2010 2011 Overall

SF

%

VT

%

Fresh

%

OR (95% CI), SF (SF [ 1) vs. VT

P value, SF vs. VT

P value

309/4,173 369/4,536 286/3,463 164/1,966 132/1,493 1,260/15,631

7.4 8.1 8.3 8.3 8.8 8.1

81/1,049 100/1,155 230/2,137 208/2,303 271/2,728 890/9,372

7.7 8.7 10.8 9.0 9.9 9.5

9,868/70,382 11,219/78,407 12,452/86,306 13,711/92,410 13,408/94,958 60,658/422,463

14.0 14.3 14.4 14.8 14.1 14.4

1.05 (0.81–1.35) 1.07 (0.85–1.35) 1.34 (1.12–1.61) 1.09 (0.88–1.35) 1.14 (0.91–1.41) 1.20 (1.09–1.31)

.727 .564 .002 .426 .248 < .001

< .001 < .001 < .001 < .001 < .001 < .001

Note: Abbreviations as in Table 2. Levi Setti. OC by slow freezing or vitrification. Fertil Steril 2014.

Beginning in 2009, the pregnancy rate per started cycle by age group was also available. As showed in Supplemental Table 1 (available online at www.fertstert.org), in the two younger age groups and in patients older than 42 years, no significant differences were found between SF and VT. Conversely, in the age group of 40–42 years the pregnancy rate was significantly higher with VT than with SF (13.8% vs. 8.5%, OR 1.72; P¼ .006). A full pregnancy outcome was available in 91% (1,686/ 1,850) of reported pregnancies, leading to 1,168 deliveries and 518 negative outcomes (30.7% of the monitored pregnancies). Pregnancies lost to follow-up were 6.4% (69/ 1,074) after SF and 12.2% (95/776) after VT procedures (P< .001). During the study period, 1,342 babies and 1,338 live babies were born from cryopreserved oocytes, 778 (8.7% per started cycle) with SF and 560 with VT (10.4% per started cycle) (P¼ .001). In total there were 11/1,388 babies (0.8%) born with major malformations, 4/778 (0.5%) with SF and 7/560 with VT (1.3%) (P¼ .145). The negative pregnancy outcome rate (spontaneous or therapeutic abortions and ectopic pregnancies) was also not significantly different between SF (324/1,005) and VT (194/681) in the evaluated years and in the overall results (32.2% vs. 28.5%, OR 0.84, 95% CI 0.68–1.04; P¼ .101). Negative outcomes analyzed for age group from 2009 to 2011 were, again, not significantly different between SF and VT (34.3% vs. 28.7%, OR 0.77, 95% CI 0.59–1; P¼ .054). To obtain one live birth with the use of the SF method a higher number of thawed oocytes is needed compared with the VT protocol (61.6 vs. 47.3, OR 1.31; Supplemental Table 2, available online at www.fertstert.org). However, it must be noted that not all of the oocytes that survived the cryopreservation/thawing or warming process were used, and no data on pregnancies after embryo cryopreservation were reported.

DISCUSSION During the years 2004–2009, embryo cryopreservation was completely banned; as a consequence, cryopreservation of oocytes was perfected and extensively used in ART programs. Italy can therefore be considered to be a pioneer (1, 2, 4, 6, 18, 21, 22) in oocyte cryopreservation. The data presented here represent the largest worldwide experience on cryopreservation and thawing/warming of supernumerary oocytes in infertile couples. In addition, this study evaluated 4

outcome from aggregated data based on either SF or VT as the method of oocyte cryopreservation. In the period 2007– 2009, SF was the procedure most commonly used, representing 81% of the cycles in 2007, 79.9% in 2008, and 61.8% in 2009 (Table 1). In the same time period the number of VT cycles grew from 568 in 2007 to 1,186 in 2009. In 2010 and 2011, VT cycles represented 55.1% and 65.6%, respectively, of the cryopreservation procedures. The total yearly numbers of CO procedures were higher in the period 2007–2009 when embryo freezing was banned. After 2009, despite the option of cryopreserving excess embryos, the number of CO procedures remained quite high, because many Italian centers were oriented toward reducing the number of transferred embryos. The data analysis showed an enormous difference in the number of cycles for each ART unit, ranging from 1 to 1,255, and revealed that not all the Italian centers offered cycles of cryopreservation, even when embryo cryopreservation was banned (16–19). In 2011, only 67% of centers reported CO cycles (Table 1). The oocyte survival rate has been consistently more efficient after vitrification (Table 2), but the fertilization rate was not significantly different. It must be remarked, however, that these results may be suffering from bias because in the period 2007–2009, when the majority of the supernumerary oocytes were cryopreserved by SF, not all of the oocytes that survived the thawing could be inseminated owing to the limit of three embryos (Table 2). In 2010 and 2011, however, when all of the survived oocytes could be inseminated, VT showed a small but statistically significant difference compared with SF in survival rate (Table 2) and implantation rate (Table 3). Our results confirm in a large sample population higher VT survival rate, higher pregnancy rate, and oocyte-to-baby rate as reported by Cil et al. in 2013 (22), but our data do not confirm higher fertilization rate. It is important to remember that our data are from infertile patients and therefore can not be compared with those reported from donor cycles (12, 23). In keeping with the concept that only a limited number of oocytes are destined to become live births per cycle (24), the observation of a low oocyte-to-baby rate is explainable by the fact that these thawed oocytes represented the supernumerary oocytes of infertile couples (and not donors) and that some oocytes from the initial cohort had already been used and produced pregnancies during the fresh cycle. VOL. - NO. - / - 2014

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1.19 (0.89–1.57) 1.21 (0.93–1.56) 1.48 (1.20–1.83) 1.03 (0.81–1.30) 1.09 (0.85–1.38) 1.26 (1.14–1.40)

.236 4.8 (0–100) 0.0 (0–50) .151 12.7 (0–100) 0.0 (0–40) < .001 8.1 (0.57–100) 0.0 (0–100) .827 8.3 (0–100) 11.1 (0–100) .508 17.6 (0–100) 11.8 (0–100) < .001 11.5 (0–50) 11.0 (0–100)

Our data do not confirm that cryopreservation of oocytes with VT gives the same results as those seen with fresh oocytes (13, 25). The main reasons for this observation is that our results represent data from infertile patients and not selected populations (such as donor oocytes), and in addition they include results from centers with minimal experience in VT. Our data represent the mean results of centers with leading experience in one or both cryopreservation protocols as well as centers with a limited number of cycles and poorer results with either SF or VT. As with other ART procedures, the results are not homogeneous among clinics and protocols, but confirm the clinical value of oocyte cryopreservation in infertile patients. Even with all of the biases, VT resulted in a statistically significant higher performance compared with SF. Our results, as the largest worldwide reported analysis, strongly support the recent American Society for Reproductive Medicine decision to remove the descriptor ‘‘experimental’’ from the OC procedure (26).

6.7 (0–40) 0.0 (0–50) 13.1 (254/1,945) 15.1 (73/483) 9.2 (0–100) 0.0 (0–53) 14.6 (312/2,136) 17.1 (90/526) 5.1 (0–50) 0.0 (0–100) 15.0 (238/1,589) 20.7 (196/946) 6.3 (0–100) 8.3 (0–50) 16.9 (151/895) 17.2 (184/1,067) 6.0 (0–100) 8.3 (0–100) 16.7 (119/711) 17.9 (233/1,301) 7.7 (0–50) 6.7 (0–100) 14.8 (1,074/7,276) 18.0 (776/4.323)

Acknowledgments: The authors thank all of the Italian ART centers that contributed with their aggregated data; without their efforts this research would not have been possible.

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1.26 (0.96–1.67) .101 1.17 (0.91–1.51) .215 1.40 (1.14–1.71) .001 0.99 (0.79–1.25) .958 1.03 (0.81–1.31) .793 1.23 (1.11–1.35) < .001

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2007 10.5 (254/2,426) 12.9 (73/568) 2008 11.9 (312/2,625) 13.7 (90/659) 2009 12.4 (238/1,916) 16.5 (196/1,186) 2010 13.8 (151/1,097) 13.7 (184/1,344) 2011 13.8 (119/863) 14.2 (233/1,644) All 12.0 (1,074/8,927) 14.4 (776/5,401)

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SUPPLEMENTAL TABLE 1 Pregnancy rates per cycle by age group following slow freezing (SF) and vitrification (VT) cycles, 2009–2011. Clinical pregnancy per cycle Age, y

Slow freezing

%34 35–39 40–42 R43 Overall

231/1,501 223/1,686 46/539 8/150 508/3,876

%

Vitrification

%

OR (95% CI), SF (SF [ 1) vs. VT

P value

15.4 13.2 8.5 5.3 13.1

274/1,737 256/1,750 76/550 7/137 613/4,174

15.8 14.6 13.8 5.1 14.7

1.03 (0.85–1.25) 1.12 (0.93–1.36) 1.72 (1.17–2.53) 0.96 (0.34–2.71) 1.14 (1.01–1.30)

.764 .236 .006 .932 .041

Note: CI ¼ confidence interval; OR ¼ odds ratio. Levi Setti. OC by slow freezing or vitrification. Fertil Steril 2014.

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ORIGINAL ARTICLE: ASSISTED REPRODUCTION

SUPPLEMENTAL TABLE 2 Thawed/warmed or fresh oocytes per live born baby following slow freezing, vitrification, and fresh cycles. Year 2007 2008 2009 2010 2011 Overall

SF

n

VT

n

Fresh

n

OR (95% CI), SF (SF [ 1) vs. VT

P value, SF vs. VT

P value

12,573/199 13,592/237 10,821/155 6,068/101 4,860/86 47,914/778

63.2 57.4 69.8 60.1 56.5 61.6

2,317/50 2,949/63 5,707/134 6,906/141 8,625/172 26,504/560

46.3 46.8 42.6 49.0 50.1 47.3

234,004/6,476 256,293/7,479 285,042/8,037 312,481/9,281 333,618/8,733 1,421,438/40,006

36.1 34.3 35.5 33.7 38.2 35.5

1.37 (1.00–1.83) 1.23 (0.93–1.63) 1.65 (1.31–2.09) 1.23 (0.95–1.59) 1.13 (0.87–1.47) 1.31 (1.17–1.46)

.047 .148 < .001 .113 .361 < .001

< .001 < .001 < .001 < .001 < .001 < .001

Note: Abbreviations as in Supplemental Table 1. Levi Setti. OC by slow freezing or vitrification. Fertil Steril 2014.

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