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Oocyte vitrification as an efficient option for elective fertility preservation

      Objective

      To provide a detailed description of the current oocyte vitrification status as a means of elective fertility preservation (EFP).

      Design

      Retrospective observational multicenter study.

      Setting

      Private university-affiliated center.

      Patient(s)

      A total of 1,468 women who underwent EFP because of age or having associated a medical condition other than cancer (January 2007 to April 2015).

      Intervention(s)

      None.

      Main Outcome Measure(s)

      Survival and cumulative live birth rate (CLBR) per consumed oocyte.

      Result(s)

      Mean age was higher with EFP due to age versus having an associated medical reason (37.7 y [95% confidence interval (CI) 36.5–37.9] vs. 35.7 y [95% CI 34.9–36.3]). In total, 137 patients (9.3%) returned to use their oocytes. Overall survival rate was 85.2% (95% CI 83.2–87.2). Live birth rate per patient was higher in women ≤35 years old than ≥36 years old (50% [95% CI 32.7–67.3] vs. 22.9% [95% CI 14.9–30.9]). CLBR was higher and increased faster in younger women. The gain in CLBR was sharp from 5 (15.4%, 95% CI −4.2 to 35.0) to 8 oocytes (40.8%, 95% CI 13.2–68.4), with an 8.4% gain per additional oocyte, in the ≤35-year-old group. The increase was slower with 10–15 oocytes, reaching a plateau CLBR of 85.2%. A milder increase (4.9% gain) was observed in the ≥36-year-old group (from 5.1% [95% CI −0.6 to 10.7] to 19.9% [95% CI 8.7–31.1] when 5–8 oocytes were consumed), reaching the plateau with 11 oocytes (CLBR 35.6%). Forty babies were born.

      Conclusion(s)

      At least 8–10 metaphase II oocytes are necessary to achieve reasonable success. Numbers should be individualized in women >36 years old. We suggest encouraging women who are motivated exclusively by a desire to postpone childbearing because of age, to come at younger ages to increase success possibilities.

      Key Words

      Discuss: You can discuss this article with its authors and with other ASRM members at http://fertstertforum.com/coboa-oocytes-vitrification-elective-fp/
      Egg banking via vitrification has proved an efficient technique in assisted reproduction (
      • Cobo A.
      • Diaz C.
      Clinical application of oocyte vitrification: a systematic review and meta-analysis of randomized controlled trials.
      ,
      • Cobo A.
      • Meseguer M.
      • Remohi J.
      • Pellicer A.
      Use of cryo-banked oocytes in an ovum donation programme: a prospective, randomized, controlled, clinical trial.
      ,
      • Rienzi L.
      • Cobo A.
      • Paffoni A.
      • Scarduelli C.
      • Capalbo A.
      • Vajta G.
      • et al.
      Consistent and predictable delivery rates after oocyte vitrification: an observational longitudinal cohort multicentric study.
      ). Currently, there are consolidated vitrification programs in assisted reproductive technology (ART) clinical practice, which has led to an increasing number of children born with the use of this technique. At present, almost 6,000 (5,842 up to December 2014) live births after the vitrification of oocytes have been accounted for in our group (unpublished data). The health of infants and the obstetrical evolution of the pregnancies conceived with vitrified oocytes are similar to those observed in our population of children conceived with fresh oocytes, thus endorsing the safety of the technique (
      • Cobo A.
      • Serra V.
      • Garrido N.
      • Olmo I.
      • Pellicer A.
      • Remohi J.
      Obstetric and perinatal outcome of babies born from vitrified oocytes.
      ). Growing evidence for the efficacy and safety of female gamete vitrification has led both the American Society for Reproductive Medicine and the European Society for Human Reproduction and Embryology to not consider this technique to be experimental (
      • Dondorp W.
      • de Wert G.
      • Pennings G.
      • Shenfield F.
      • Devroey P.
      • Tarlatzis B.
      • et al.
      Oocyte cryopreservation for age-related fertility loss.
      ,
      Mature oocyte cryopreservation: a guideline.
      ).
      For these reasons, vitrification of oocytes is currently being offered as an option for women who wish to preserve their gametes to allow them to have a chance to conceive in the future and to have their own genetic offspring (
      • Cobo A.
      • Garcia-Velasco J.A.
      • Domingo J.
      • Remohi J.
      • Pellicer A.
      Is vitrification of oocytes useful for fertility preservation for age-related fertility decline and in cancer patients?.
      ). The main beneficiaries of this strategy are cancer patients who must undergo chemotherapy or radiotherapy and patients with other diseases who require potentially gonadotoxic treatments. Ovarian tissue cryopreservation has been applied to cancer patients for fertility preservation (FP) purposes. Another population to benefit from FP is made up of those women who wish to postpone childbearing for various reasons usually known as “social reasons.” The biggest threat in these cases is age, which is why this indication is also known as elective FP (EFP) due to age-related fertility decline (
      • Dondorp W.
      • de Wert G.
      • Pennings G.
      • Shenfield F.
      • Devroey P.
      • Tarlatzis B.
      • et al.
      Oocyte cryopreservation for age-related fertility loss.
      ). EFP can also help women to endure some medical conditions other than cancer that can trouble their future fertility, such as endometriosis or other alterations that lead to premature menopause. In these cases, the condition itself is not an impediment to getting pregnant at the time of diagnosis, but, for a variety of reasons, they decide to postpone motherhood; therefore, they choose to vitrify their oocytes for future IVF treatments. In a significant proportion of cases, they are diagnosed when planning the vitrification cycle, or if previously diagnosed, they go on with the EFP after being counseled by their specialist. On the other hand, there are other medical conditions that contraindicate pregnancy at the time of diagnosis, such as some disorders that require potentially iatrogenic treatments, e.g., autoimmune diseases, or even a clinical situation that entails having to undergo bilateral ovariectomy. EFP is strongly advisable in these cases.
      Currently, assisted reproduction centers are noticing a considerable increase in the demand for EFP requests for all the above reasons. All of the women seek a “time-out” until they reach the right time to try to get pregnant. In many cases they need to overcome particular situations, including lack of a partner, need to solve financial or career issues, and many others. Solving these particular situations usually implies that they need to take their time, which means that an undetermined time period will pass before they decide to come back to use their oocytes. Given these particuliarities, knowledge of the women's real possibilities is still lacking. Therefore, they have sought counseling about their possibilities based on assumptions that originate from outcomes achieved in other populations, namely, oocyte recipients and other infertile patients, after oocyte vitrification.
      With the present study, we aimed to provide a detailed description of the current situation of EFP in our group, including the profile of the woman who vitrified oocytes, the description of the rate at which they return to use their oocytes, their clinical outcomes and the probability of having a baby according to the number of oocytes consumed. This study provides data on the outcomes attained after EFP via oocyte vitrification, achieved in actual FP patients to avoid extrapolations from other populations. This information will be most welcome, because evidence on this matter is lacking.

      Materials and methods

       Study Design and Study Population

      A retrospective multicenter study included all of the women (n = 1,468) who electively vitrified their oocytes for FP purposes because of age or having an associated medical condition other than cancer from January 2007 to April 2015 at 13 different Spanish clinics from our group. Institutional Review Board approval was obtained (1505-VLC-033-AC). Data were collected from computerized clinical charts and were anonymized according to Spanish law on assisted reproduction (Law 14/2007 on Biomedical Research). The vast majority of the included women (n = 1,382) opted for EFP because of age-related fertility decline (social reasons). The reason the remaining women (n = 86) chose FP was the presence of a medical condition other than cancer that could undermine future fertility, such as endometriosis or low ovarian reserve. Overall, 137 women returned to use their oocytes. A small group of six patients (seven controlled ovarian stimulation [COS] cycles) with a disease that required a potentially iatrogenic treatment or on a therapy that discouraged gestation during the medical management time also vitrified oocytes for FP during the study period time. The diseases involved were transverse myelitis, thyroid nodule, and multiple sclerosis (the latter patient also needed bilateral ovariectomy owing to the presence of teratoma). None of these patients had returned to use their oocytes at the time of writing.

       Controlled Ovarian Stimulation Protocol

      COS was initiated on day 2 or 3 of a spontaneous cycle. An initial dose of 225–300 IU recombinant FSH (rFSH; Gonal-F [Merck-Serono] or Puregon [MSD]) and/or highly purified hMG (Menopur; Ferring Pharmaceuticals) was administered. From day 6 onward, the gonadotropin dose was estimated according to serum E2 levels and a transvaginal ultrasound scan. When a leading follicle reached 13–14 mm, a GnRH antagonist (Cetrotide [Merck-Serono] or Orgalutran [MSD]) was administered at 0.25 μg/d. Final oocyte maturation was triggered with the use of 250 μg recombinant hCG (rhCG; Ovitrelle [Merck-Serono]) as soon as the mean diameters of two follicles were ≥18 mm. In some cases, triggering was performed with a single dose of a GnRH agonist (0.1 mg triptorelin [Decapeptyl; Ipsen Pharma]). Oocyte retrieval was scheduled 36 hours after hCG injection.

       Oocyte Vitrification and Warming

      Oocytes were denuded 2 hours after oocyte retrieval. Following the nuclear maturity evaluation, only the metaphase II (MII) oocytes were selected for immediate vitrification. All the vitrification and warming solutions were obtained from Kitazato. The Cryotop method used for oocyte vitrification has been described elsewhere (
      • Cobo A.
      • Meseguer M.
      • Remohi J.
      • Pellicer A.
      Use of cryo-banked oocytes in an ovum donation programme: a prospective, randomized, controlled, clinical trial.
      ). Briefly, after 12 minutes of stepwise equilibration in a mixture of 15% (v/v) ethylene glycol + dimethylsulfoxide, oocytes were subjected to a vitrification solution for 50–60 seconds that contained 30% (v/v) of the same cryoprotectants. Hydroxypropyl cellulose, as a substitute for synthetic serum substitute, and trehalose, as a substitute for sucrose, were also used in the solutions for vitrification and warming. No statistical differences were found in either survival or clinical outcomes when the differential use of these additives was tested (data not shown). The preliminary study results have been previously revealed (
      • Coello A.
      • Campos P.
      • Remohí J.
      • Meseguer M.
      • Cobo A.
      A combination of hydroxypropyl cellulose and trehalose as supplementation for vitrification of human oocytes: a retrospective cohort study.
      ). Loading took place within the next 10 seconds by placing the oocytes contained in a minimum volume on the device. Vitrification was induced by immediate plunging into liquid nitrogen. Four oocytes (maximum) were loaded per Cryotop. Oocytes were stored in vapor tanks (V1500-AB Isothermal Freezer; CBS) for a variable storage time (
      • Cobo A.
      • Romero J.L.
      • Perez S.
      • de los Santos M.J.
      • Meseguer M.
      • Remohi J.
      Storage of human oocytes in the vapor phase of nitrogen.
      ). During warming, cryoprotectants were diluted by subjecting oocytes to a hyperosmolar solution that contained 1.0 mol/L sucrose or trehalose at 37°C. Dilution continued for 3 minutes at room temperature in a half-diluted solution that contained the same sugar. The warming procedure finished with two washes in buffer solution at room temperature for 5 minutes and 1 minute, respectively. The vitrification and warming procedures of the surplus embryos were performed as explained in detail elsewhere (
      • Cobo A.
      • de los Santos M.J.
      • Castello D.
      • Gamiz P.
      • Campos P.
      • Remohi J.
      Outcomes of vitrified early cleavage-stage and blastocyst-stage embryos in a cryopreservation program: evaluation of 3,150 warming cycles.
      ). The procedures were similar to those described above for oocytes, except that equilibration was performed in a single step (not step-wise) for ∼10 minutes. All of the remaining procedures (vitrification step, loading storage, and warming/dilution) were carried out as described for oocytes.

       IVF Procedures

      The surviving oocytes were placed under standard culture conditions at 37°C in 5.5% CO2 and at room atmospheric oxygen concentration (20% O2) for 2 hours before intracytoplasmic sperm injection (ICSI). All of the embryos were cultured under the same conditions in Cleavage Medium (Cook IVF) until day 3, and in CCM Medium (Vitrolife) from days 3 5–6 for blastocyst embryo transfer (ET).

       Endometrial Preparation for Embryo Transfer

      The endometrial preparation protocol has been described elsewhere (
      • Soares S.R.
      • Troncoso C.
      • Bosch E.
      • Serra V.
      • Simon C.
      • Remohi J.
      • et al.
      Age and uterine receptiveness: predicting the outcome of oocyte donation cycles.
      ). After menses, all subjects received oral estradiol valerate (EV; Progynova, 6 mg/d; Schering). Approximately 10 days after initiating EV, serum E2 levels and endometrial thickness were measured. Administration of micronized progesterone (P; 800 mg/d, vaginally; Progeffik) was initiated 2 days before ET if it was a day 3 ET. For blastocyst ET, P was initiated 4 days before the transfer. If pregnancy was achieved, administration of EV and P was maintained until gestation week 12. ET was scheduled usually 4–8 hours after warming either for day 3 embryos or for blastocysts.

       Statistical Analysis

      The main outcome measures were survival rate, pregnancy rate, and cumulative live birth rate (CLBR). Secondary outcomes were age at vitrification, number of oocytes retrieved and vitrified according to patient age, storage time, return rate, and clinical and ongoing pregnancy rate.
      Survival was morphologically confirmed after warming by the appearance of oocytes presenting cytoplasm refringence and integrity of the oolemma and the zona pellucida. Biochemical pregnancy was assumed with positive β-hCG levels on day 14, as was the clinical outcome analysis according to day 3 versus blastocyst-stage ET. Clinical pregnancy was confirmed when an intrauterine gestational sac was revealed in a transvaginal ultrasound scan ≥5 weeks after ET (
      • Zegers-Hochschild F.
      • Adamson G.D.
      • de Mouzon J.
      • Ishihara O.
      • Mansour R.
      • Nygren K.
      • et al.
      International Committee for Monitoring Assisted Reproductive Technology (ICMART) and the World Health Organization (WHO) revised glossary of ART terminology, 2009.
      ). Ongoing pregnancy was defined as clinical pregnancy with fetal heart beat (FHB) at ≥12 weeks (
      • Bonduelle M.
      • Liebaers I.
      • Deketelaere V.
      • Derde M.P.
      • Camus M.
      • Devroey P.
      • et al.
      Neonatal data on a cohort of 2889 infants born after ICSI (1991–1999) and of 2995 infants born after IVF (1983–1999).
      ), and the implantation rate (with FHB) was deferred to implantation per transferred embryo. Delivery was confirmed when a patient gave birth to a live child at or beyond gestation week 24.
      Chi-square tests were used to compare categoric data, and t tests or analyses of variance with post hoc comparisons (Bonferroni) were used to compare the means within groups whenever appropriate. A P value of <.05 was considered to be significant. Statistical analyses were performed with the use of SPSS software (version 19.0).
      CLBR was estimated by means of the Kaplan-Meier method according to the total number of oocytes consumed in consecutive procedures, including those that resulted in cancelled embryo transfers, and those from both fresh or frozen embryo transfers, if this was the case, until a live birth was achieved, as previously validated (
      • Garrido N.
      • Bellver J.
      • Remohi J.
      • Simon C.
      • Pellicer A.
      Cumulative live-birth rates per total number of embryos needed to reach newborn in consecutive in vitro fertilization (IVF) cycles: a new approach to measuring the likelihood of IVF success.
      ). All of the patients who used their oocytes were counted in this analysis, including those who consumed oocytes and did not get pregnant, to compute all of the oocytes. The term “consumed oocyte” involved all those that survived or did not, those that were fertilized or not, and those that developed to usable embryos or were arrested. The embryos that were cryopreserved and had not yet been used were excluded from the analysis. Data were further stratified and analyzed according to age with the use of the log-rank, Breslow, and Tarone-Ware tests to compare survival curves according to each case.

      Results

       Demographics

      Increasing numbers of EFP cycles have been performed in our centers over the years. Thus in 2015, this figure had multiplied fivefold compared with the number of cycles performed in 2007 when we initiated our FP program (Supplemental Fig. 1, available online at www.fertstert.org). Currently, the proportion of EFP corresponds to ∼25% of all the oocyte vitrification cycles in our group (Supplemental Fig. 1). In all, 18,915 MII oocytes collected from 1,468 women who completed 2,137 (mean 1.5, 95% CI 1.4–1.7) COS cycles were included in the study (Table 1). Supplemental Figure 2 (available online at www.fertstert.org) shows the distribution of EFP cycles. The vast majority (94.2%) were motivated by age-related issues, whereas the reason in 2.1% was the presence of endometriosis, 1.7% were due to low ovarian reserve, and 1.6% needed ovarian surgery. Other reasons were behind the remaining 0.5%. The majority of women were single heterosexual (75.6%), 23.9% were patients within heterosexual relationships, whereas a very small minority were homosexual women (0.4%; Supplemental Fig. 3, available online at www.fertstert.org). They mostly reported a high level of education (72.8%; Supplemental Fig. 3). Mean age at vitrification was 37.2 years (95% CI 36.9–37.3) (Table 1). As shown in Supplemental Figure 4A (available online at www.fertstert.org), in the group of EFP due to age, most women came to vitrify at ages 37–39 years (63%), 18.9% were 31–35 years, and 16.2% were aged ≥40 years at the time of vitrification. The remaining minority were <30 years. Conversely, most women who vitrified because of a concurrent medical condition did it at the ages of 31–35 years (33.1%) and 36–39 years (29.6%). Nearly 30% (18.9% + 10.7%) did it when they were <30 years of age (Supplemental Fig. 4B). Only 7.7% of women vitrified at ≥40 years of age in this group. Age at vitrification was higher in the group of EFP due to age (37.7 y [95% CI 37.5–37.9] vs. 35.7 y [95% CI 34.9–36.3]; Supplemental Table 3, available online at www.fertstert.org).
      Table 1Baseline characteristics, COS parameters, IVF data, and overall clinical outcomes.
      ParameterMean95% CI
      Baseline characteristics
       Patients, n1,468NA
       No. of cycles (per patient)2,137 (1.5)1.4–1.7
       Age at FP, y37.236.9–37.3
       Patients with previous children, n (%)18 (1.4)0.8–2.0
      Controlled ovarian stimulation (COS)
       Length of stimulation10.97 ± 1.710.9–11.1
       Total FSH dose (IU)1,308.8 ± 670.81,271.8–1,345.8
       Total hMG dose (IU)786.4 ± 705.7747.5–825.3
       E2 on day of hCG (pg/mL)1,511.7 ± 1,172.561,447.1–1,576.3
      IVF
       No. of retrieved oocytes (per patient)18,915 (12.9)12.5–13.3
       No. of retrieved oocyte (per cycle)18,915 (8.9)8.6–9.2
       No. of MII oocytes vitrified (per patient)14,415 (9.8)9.5–10.2
       No. of MII oocytes vitrified (per cycle)14,415 (6.7)6.5–7.1
       Patients with no egg retrieval, n (%)7/2,137 (0.3)0.1–0.7
       COS cycles with no egg retrieval, n (%)12/2,137 (0.6)0.1–0.9
      Clinical outcomes
       No. of patients returning to use their oocytes137NA
       Age when returning, y39.239.0–40.1
       No. of oocyte warming cycles (per patient)148 (1.1)1.0–1.2
       No. of MII oocytes warmed (per patient)1,182 (8.6)8.3–8.9
       Mean storage time, y2.11.9–2.5
       Survival rate, n (%)1,007/1,182 (85.2)83.2–87.2
       ETs/patients at 1st warming, n (%)108/137 (78.8)71.7–86.1
       ETs/patients (1st + 2nd warming cycles
      137 1st warming cycles + 11 2nd or 3rd warming cycles.
      ), n (%)
      117/137 (85.4)80.2–92.2
       No. of embryos transferred (per patient)181 (1.5)14–1.6
       Implantation rate, %35.928.9–42.9
       CPR/transfer, n (%)54/117 (46.2)33.9–52.8
       OPR/transfer, n (%)37/117 (31.6)20.6–37.9
       CPR/patient, n (%)54/137 (39.4)29.6–47.8
       OPR/patient (%)37/137 (27.0)23.1–39.8
       CPR/COS cycle, n (%)54/148 (36.5)27.1–42.1
       OPR/COS cycle, n (%)37/148 (25)18.1–31.5
       Deliveries26
      11 other pregnancies still ongoing.
      NA
       No. of live births31
      Three sets of twins.
      NA
      Note: CI = confidence interval; CPR = clinical pregnancy rate; ET = embryo transfer; FP = fertility preservation; MII = metaphase II; OPR = ongoing pregnancy rate.
      a 137 1st warming cycles + 11 2nd or 3rd warming cycles.
      b 11 other pregnancies still ongoing.
      c Three sets of twins.

       Baseline Characteristics and IVF Data

      Table 1 presents the data baseline characteristics, COS cycle parameters, and IVF data in the overall population. Mean numbers of 9.8 oocytes (95% CI 9.5–10.2) per patient and 6.7 (95% CI 6.5–7.1) per cycle were vitrified. There were a few cases in which no oocytes were retrieved (Table 1). Figure 1 shows the number of retrieved oocytes (including germinal vesicle, metaphase I, MII, and atresic oocytes) and the number of MII finally vitrified according to the different patients' age groups. Means of 8.8 (95% CI 8.40–9.51) and 6.6 (95% CI 5.8–6.9) oocytes were retrieved and vitrified at ages 36–40 years. These figures were 5.1 (95% CI 4.2–6.0) and 3.9 (95% CI 2.6–5.0) for patients >40 years of age (Fig. 1). Statistical differences were observed in both cases compared with the younger age groups (P<.05).
      Figure thumbnail gr1
      Figure 1Distribution of retrieved and vitrified metaphase II (MII) oocytes according to women's ages (years). Different superscripts indicate statistical differences between groups (P<.05).

       Clinical Outcome

      One hundred thirty-seven patients returned to use their oocytes (return rate 9.3%, 95% CI 7.8–10.8) at a mean age of 39.2 years (95% CI 39.0–40.1) and after a mean storage period time of 2.1 years (95% CI 1.9–2.5; Table 1). Of these, 120 patients had electively vitrified their oocytes due to age, and the remaining 17 had had an associated medical condition (12 patients had endometriosis, three underwent ovarian surgery, and 2 had low ovarian reserve at the time of vitrification). Supplemental Table 1 presents the distribution of single women and women with partners at vitrification and their status when returning. Of the 120 patients who vitrified due to age, 79.2% were single at vitrification; almost one-half of them had a partner when they returned to use their oocytes (47.4%). The 41.7% who remained single used donor sperm in their IVF cycle. In the small group of patients who vitrified owing to a medical reason and returned, the great majority had a partner at the time of vitrification (76.5%), and, interestingly, none remained single when they returned. Supplemental Figure 5 (available online at www.fertstert.org) presents the distribution of warming cycles by the age at vitrification and the mean storage time, according to the reason for FP. The majority of women who returned in the group of EFP due to age, stored their gametes at the age of 36–40 years and returned 2.2 years later to use their oocytes (Supplemental Fig. 5A). Most women who electively stored oocytes because of a concurrent medical condition did it younger, at the ages of 31–35 years, and returned 1.4 years later. In this group, the youngest women waited longer to return (4.0 y; Supplemental Fig. 5B).
      We avoided warming up the entire number of oocytes stored by a patient at once to save some MII oocytes for future attempts. However, sometimes we had to warm more oocytes from some patients owing to adverse outcomes after the first IVF attempt. Accordingly, 148 warming cycles (mean 1.1 per patient, 95% CI 1.0–1.2), were performed (137 at first warming and 11 after a second or third warming; Table 1). A total of 1,182 oocytes were warmed up (mean 8.6 per patient, 95% CI 8.3–8.9). A number of 117 (mean per patient 85.4%, 95% CI 80.2–92.2) ETs were performed. The overall survival rate was 85.2% (95% CI 83.2–87.2). Day 3 ET was performed with 86 of them, and blastocysts were transferred to 31 patients. Twenty-six patients among those who vitrified due to age were subjected to preimplantation genetic screening (PGS; mean age 42.1 y; 95% CI 41.3–44.3). Fifteen ETs were cancelled owing to the absence of chromosomally normal embryos. The clinical and ongoing pregnancy rates per patient were, respectively, 39.4% (95% CI 29.6–47.8) and 27.0% (95% CI 23.1–39.8) (Table 1).
      Thirty-one babies were born by the time of writing, and 11 pregnancies are still ongoing. Surplus embryos were vitrified by 62 patients (Supplemental Table 2, available online at www.fertstert.org). Forty-one cryotransfers have been performed to date with 33 patients (26 day 3 ETs and 15 blastocyst ETs). Nine other babies have been born. The cumulative clinical and ongoing pregnancy rates when three ETs were completed (1 fresh ET + 2 cryotransfers) were 73.4% and 59.6%, respectively. When considering the number of babies born after fresh ETs and cryotransfers, 40 children were born in total, with a mean birth weight of 2,903 ± 1,058 g, of whom 59% were male. One female baby died owing to prematurity.
      The clinical outcome is also presented according to the reason for FP (Supplemental Table 3, available online at www.fertstert.org). Statistical differences were observed in mean age, number of oocytes, return rate, and survival and clinical outcomes, showing lower success rates in the group of EFP due to age; however, the number of patients analyzed was very low in the group of EFP with an associated medical condition.
      A different analysis of survival and clinical outcomes was made by categorization of age at vitrification according to different groups. Table 2 first presents the outcomes according to two categorizations: women aged ≤35 versus ≥36 years. Survival was higher in the younger group (94.6% [95% CI 91.9–97.3] vs. 82.4% [95% CI 79.9–84.9]). The live birth rate per patient was statistically higher in the younger compared with the older patients (50% [95% CI 32.7–67.3] vs. 22.9% [95% CI 14.9–30.9]). Table 2 then presents the outcomes according to different subgroups of age, showing the noticeable decrease in live birth rate from the youngest category of women aged ≤29 years (100% [95% CI 100–100]) to the oldest group of women aged 40–44 years (3.7% [95% CI −3.4 to 10.8]). Notably, because these were the extreme categories, the groups contained a very low number of patients as indicated by the wide-ranging 95% CI. Nonetheless, the strong effect of the age is clearly revealed in this analysis.
      Table 2Survival and clinical outcomes according to age at time of vitrification, n (%).
      Age, yPatients, nCycles, nSurvival rate, n (%)CPR/cycle, n (%)CPR/ET, n (%)OPR/cycle, n (%)OPR/ET, n (%)Live births/patients, n (%)
      Survival and clinical outcomes in patients aged ≤35 y and ≥36 y at vitrification
       ≤353241257/272 (94.6)
      Different superscripts in the same column indicate statistical differences (P<.05).
      24/41 (58.5)
      Different superscripts in the same column indicate statistical differences (P<.05).
      24/39 (61.5)
      Different superscripts in the same column indicate statistical differences (P<.05).
      21/41 (51.2)
      Different superscripts in the same column indicate statistical differences (P<.05).
      21/39 (53.9)
      Different superscripts in the same column indicate statistical differences (P<.05).
      16/32 (50)
      Different superscripts in the same column indicate statistical differences (P<.05).
       ≥36105150750/910 (82.4)
      Different superscripts in the same column indicate statistical differences (P<.05).
      47/150 (31.3)
      Different superscripts in the same column indicate statistical differences (P<.05).
      47/118 (39.8)
      Different superscripts in the same column indicate statistical differences (P<.05).
      27/150 (18.0)
      Different superscripts in the same column indicate statistical differences (P<.05).
      27/118 (22.9)
      Different superscripts in the same column indicate statistical differences (P<.05).
      24/105 (22.9)
      Different superscripts in the same column indicate statistical differences (P<.05).
       Total1371911,007/1,182 (85.2)71/191 (37.1)71/157 (45.2)48/191 (25.1)48/157 (30.5)40/137 (29.2)
      Survival and clinical outcomes according to different groups of age at vitrification
       ≤296959/62 (94.5)
      Different superscripts in the same column indicate statistical differences (P<.05).
      6/9 (66.6)
      Different superscripts in the same column indicate statistical differences (P<.05).
      6/9 (66.6)
      Different superscripts in the same column indicate statistical differences (P<.05).
      6/9 (66.6)
      Different superscripts in the same column indicate statistical differences (P<.05).
      6/9 (66.6)
      Different superscripts in the same column indicate statistical differences (P<.05).
      6/6 (100)
      Different superscripts in the same column indicate statistical differences (P<.05).
       30–342023155/161 (96.1)
      Different superscripts in the same column indicate statistical differences (P<.05).
      14/23 (60.9)
      Different superscripts in the same column indicate statistical differences (P<.05).
      14/21 (66.7)
      Different superscripts in the same column indicate statistical differences (P<.05).
      13/23 (56.5)
      Different superscripts in the same column indicate statistical differences (P<.05).
      13/21 (61.9)
      Different superscripts in the same column indicate statistical differences (P<.05).
      9/20 (45)
      Different superscripts in the same column indicate statistical differences (P<.05).
       35–3984127601/734 (81.8)
      Different superscripts in the same column indicate statistical differences (P<.05).
      48/127 (37.8)
      Different superscripts in the same column indicate statistical differences (P<.05).
      48/112 (42.9)
      Different superscripts in the same column indicate statistical differences (P<.05).
      27/127 (21.3)
      Different superscripts in the same column indicate statistical differences (P<.05).
      27/112 (24.1)
      Different superscripts in the same column indicate statistical differences (P<.05).
      24/84 (28.5)
      Different superscripts in the same column indicate statistical differences (P<.05).
       ≥402732192/225 (85.3)
      Different superscripts in the same column indicate statistical differences (P<.05).
      3/32 (9.8)
      Different superscripts in the same column indicate statistical differences (P<.05).
      3/15 (20)
      Different superscripts in the same column indicate statistical differences (P<.05).
      2/32 (6.3)
      Different superscripts in the same column indicate statistical differences (P<.05).
      2/15 (13.3)
      Different superscripts in the same column indicate statistical differences (P<.05).
      1 (3.7)
      Different superscripts in the same column indicate statistical differences (P<.05).
       Total1371911,007/1,182 (85.2)71/191 (37.1)71/157 (45.2)48/191 (25.1)48/157 (30.5)40/137 (29.2)
      Note: Abbreviations as in Table 1.
      a,b,c Different superscripts in the same column indicate statistical differences (P<.05).
      The CLBR is shown in Figure 2 according to the number of oocytes consumed. Figure 2 shows the Kaplan-Meier survival curves according to patient's age. There is a clear different probability of having a baby according to the number of oocytes consumed when the ≤35-year-old and ≥36-year-old groups are compared (P<.05). The CLBR in patients aged ≤35 years was significantly higher compared with those aged ≥36 years, despite using the same number of oocytes. For example, the probability of live birth for the patients aged ≤35 years for whom eight oocytes were used was 40.8% (Fig. 2), whereas it was statistically lower (19.9% when eight oocytes were used; P<.05) for the patients aged ≥36 years. On the other hand, the increase in CLBR occurred very rapidly from five to eight oocytes in the group of ≤35-year-old women, and the increase was much slower in the ≥36-year-old group. The CLBR with five oocytes was 15.4% and was 40.8% with eight oocytes, so the difference was 25.4%, which was gained with the use of only three additional oocytes. Thus, within this range of the curve, the “rhythm” or pace at which CLBR increased was 25.4/3. This implies an 8.4% gain per additional oocyte. The pace of gain became slightly slower from 10 to 15 oocytes, when a plateau was reached, with a CLBR of 85.2% (Fig. 2). In the ≥36-year-old group the CLBR with five oocytes was 5.1% and with eight oocytes was 19.9%, so the difference was 14.8%, meaning 4.9% (14.8/3) gain in this part of the curve. The gain became much slower per additional oocyte, reaching the plateau (CLBR 35.6%) with 11 oocytes in this age group, indicating that success was not increased regardless of the increase in the number of oocytes consumed in this group.
      Figure thumbnail gr2
      Figure 2Kaplan-Meier plotting of the cumulative live birth rates (CLBR) of at least one baby, depending on the total number of consumed oocytes and categorized by age (≤35 y and ≥36 y). Overall comparisons: log rank (Mantel-Cox); P=.003; Tarone-Ware; P=.011. The table shows the CLBRs and 95% confidence intervals (CsI) when 5–15 oocytes were consumed, according to age.

      Discussion

      The major advancements made with vitrification in the past few years have made egg banking a new developing area in ART. The first patients to consider oocyte freezing were cancer patients who wanted to avoid the ethical concerns of creating embryos after being diagnosed with cancer and/or with no partner. Recently, the demand for oocyte cryopreservation for nononcologic indications has dramatically increased and has reached social media and society (

      Rosenblum E. Later, baby: will freezing your eggs free your career. Available at: http://www.bloomberg.com/bw/articles/2014-04-17/new-egg-freezing-technology-eases-womens-career-family-angst. Last accessed December 22, 2015.

      ). We herein described how this demand has increased fivefold in the past 8 years at our centers. We also analyzed those factors that could contribute to success, which revealed data that could help us adequately counsel our patients.
      The main group of patients who demand egg freezing for nonmedical indications comprises those at risk of reduced reproductive capacity due to age-related fertility decline. This is an increasingly common indication in ART units. In fact, the demand for ART has increased 9% from 2003 to 2009, but this rise sores to 41% in women older than 40 years (

      National Center for Chronic Disease Prevention and Health Promotion. Assisted reproductive technology. National Summary Report 2013. Available at: http://www.cdc.gov/art/pdf/2013-report/art_2013_national_summary_report.pdf#page=11. Accesed October 20, 2015.

      ). Similarly, maternal age of first pregnancy has continuously increased since the 1970s (

      Instituto Nacional de Estadística. Edad Media a la Maternidad por orden del nacimiento según nacionalidad (española/extranjera) de la madre. Available at: http://www.ine.es/jaxiT3/Datos.htm?t=1579. Accessed September 9, 2015.

      ). The reasons for this are complex and varied, but the sociocultural environment undoubtedly drives women to look for personal, professional, and economic stability before embarking on having a child. In our series, almost 80% of our patients were single at the time of oocyte freezing, and ∼75% of these women had a high level of education. As described by other authors (
      • Dondorp W.
      • de Wert G.
      • Pennings G.
      • Shenfield F.
      • Devroey P.
      • Tarlatzis B.
      • et al.
      Oocyte cryopreservation for age-related fertility loss.
      ,
      • Lockwood G.M.
      Social egg freezing: the prospect of reproductive “immortality” or a dangerous delusion?.
      ,
      • Hodes-Wertz B.
      • Druckenmiller S.
      • Smith M.
      • Noyes N.
      What do reproductive-age women who undergo oocyte cryopreservation think about the process as a means to preserve fertility?.
      ), the difficulty of finding the appropriate partner and lack of couples' compromise to create a family are two of the main reasons for postponing maternity, especially in these highly educated women.
      Given the impact of social changes on lifestyle, an increasingly large group of women who share various demographic characteristics has emerged: they are usually aged ≥40 years, typically heterosexual, often highly skilled, and confident, and financially independent, but they have no steady partner. These “new” single women live alone and have been forced to build new social networks and to develop new activities to generate their own personal spaces. This minor social interaction further reduces the chances of finding a partner. It should also be mentioned that remaining single is a free choice for many women, because nowadays most prefer to be alone than living with someone who does not live up to their expectations. In any case, this status favors delaying having a family and motherhood. For many single women who are getting older, the biologic clock represents a serious threat that jeopardizes the possibility of having a biologic child. Usually, the thought of “running out of time” is a source of a great deal of pressure for them, which is often accompanied by an extra dose of social pressure and criticism from their own environment. Oocyte freezing offers an excellent opportunity to alleviate this pressure while, and if, the right partner appears. A recent survey has provided good evidence to help better understand the motivations of women who seek oocyte cryopreservation as a means of FP (
      • Hodes-Wertz B.
      • Druckenmiller S.
      • Smith M.
      • Noyes N.
      What do reproductive-age women who undergo oocyte cryopreservation think about the process as a means to preserve fertility?.
      ). This survey showed that women who have undergone cryopreservation are well aware of fertility decline due to age but continue to delay child bearing because of their own individual circumstances. Furthermore, the authors concluded that “oocyte cryopreservation may increase a woman's security regarding her ability to bear her own genetic offspring,” which explains why oocyte cryopreservation is gaining popularity.
      In the past few years, the demand for ART in women >40 years old has noticeably grown (
      • Lockwood G.M.
      Social egg freezing: the prospect of reproductive “immortality” or a dangerous delusion?.
      ). We are witnessing not only an increasing delay in the age of first pregnancy, but also in the number of children born in women >35 years old. This fact highlights the potential benefits of oocyte vitrification, because FP will be higher at younger ages for different reasons: 1) more chances of success; 2) fewer unsuccessful IVF cycles due to advanced maternal age, with the emotional and economical frustration that they generate; and 3) less need for donor oocytes if women bank their own eggs (
      • Lockwood G.M.
      Social egg freezing: the prospect of reproductive “immortality” or a dangerous delusion?.
      ,
      • Mertes H.
      • Pennings G.
      Social egg freezing: for better, not for worse.
      ). However, our data show that women currently still consult at a very late stage. The fact that the patients in our series consulted at the age of 37 years reflects this reality, which is beyond the optimal age for ART. Thus, elective freezing of oocytes for nonmedical indications is becoming one of the most frequent indications for fertility preservation, but still in a late stage.
      It is worth mentioning that in our series some women had an associated medical condition when they decided on EFP. Most of them came to our clinics, motivated by the desire for FP, however, during the preparation of the cycle for oocyte vitrification, an as yet undiagnosed medical condition related to infertility, such as endometriosis or low ovarian reserve, was identified. In contrast, other women were diagnosed before seeking FP, as in the cases of the need of bilateral ovariectomy or when a potentially gonadotoxic treatment was required. In our series, these were the great minority, and all of them electively vitrified their gametes.
      When discussing the efficacy of oocyte freezing, it is crucial to not generate unrealistic expectations in patients. Most survival data after vitrification and thawing come from donor oocytes, which are from very young women and have survival rates of >95% (
      • Cobo A.
      • Meseguer M.
      • Remohi J.
      • Pellicer A.
      Use of cryo-banked oocytes in an ovum donation programme: a prospective, randomized, controlled, clinical trial.
      ,
      • Nagy Z.P.
      • Chang C.C.
      • Shapiro D.B.
      • Bernal D.P.
      • Elsner C.W.
      • Mitchell-Leef D.
      • et al.
      Clinical evaluation of the efficiency of an oocyte donation program using egg cryo-banking.
      ,
      • Garcia J.I.
      • Noriega-Portella L.
      • Noriega-Hoces L.
      Efficacy of oocyte vitrification combined with blastocyst stage transfer in an egg donation program.
      ). However, we now know that this is not the case for older patients, and most women who still come for FP today are aged >35 years. It is well known that IVF outcomes worsen from the age of 35 years onward, and becomes much worse at ≥40 years. As shown by the European Registry on Assisted Reproductive Technologies, pregnancy and delivery rates at the age of 35–39 years are 26.6% and 18.5%, respectively, and drop to 14% and 7.7% in women aged >40 years (
      • Kupka M.S.
      • Ferraretti A.P.
      • de Mouzon J.
      • Erb K.
      • d'Hooghe T.
      • Castilla J.A.
      • et al.
      Assisted reproductive technology in Europe, 2010: results generated from European registers by ESHRE.
      ). It should be mentioned that so far, the outcomes reported in the registries consists primarily in results achieved with fresh oocytes. Our findings from the present study correlate with these outcomes, thus endorsing the validity of the use of vitrified oocytes in assisted reproduction. Likewise, the efficacy of oocyte vitrification as a part of the infertile treatment was previously demonstrated in a multicentric study with the use of vitrified autologous oocytes (
      • Rienzi L.
      • Cobo A.
      • Paffoni A.
      • Scarduelli C.
      • Capalbo A.
      • Vajta G.
      • et al.
      Consistent and predictable delivery rates after oocyte vitrification: an observational longitudinal cohort multicentric study.
      ).
      We have reported here, in the largest series to date, our experience in FP in a population of women who electively decided to vitrify their gametes for future use. A clear, and expected, effect of female age was observed in our data. Higher outcomes were achieved in women aged ≤35 years. In this group a larger number of oocytes were retrieved and finally vitrified, and the survival and clinical outcomes were equivalent to those achieved in our egg-banking program for ovum donation, with the highest success rates in the youngest group of women (≤29 years). Otherwise, predictably fewer oocytes and worse outcomes were achieved as the age increased, resembling the results of the infertile population of similar age. These observations were confirmed when the analysis was made according to the reason for EFP. According to our data, the women who electively vitrified due to age were older than those who did it with an associated medical condition, a fact that had a negative impact on the survival and clinical outcomes, although it should be noted that the number of patients in the second group was very low (n = 17).
      Another way of looking at oocyte freezing efficiency is by modeling. Van Loendersloot et al. (
      • van Loendersloot L.L.
      • Moolenaar L.M.
      • Mol B.W.
      • Repping S.
      • van der Veen F.
      • Goddijn M.
      Expanding reproductive lifespan: a cost-effectiveness study on oocyte freezing.
      ) demonstrated with a very elegant model how oocyte freezing was significantly more cost-effective. Three scenarios for a women aged 35 years, who decided to have babies at the age of 40 years, were suggested. Strategy 1 was to do three oocyte freezing cycles, and then to use the frozen eggs for IVF at 40 years; if unsuccessful, attempt spontaneous pregnancy until 45 years. Strategy 2 consisted in not doing anything, and then attempting spontaneous pregnancy at 40–45 years. Strategy 3 involved waiting until the age of 40 years and then doing three IVF cycles; then if unsuccessful, attempt spontaneous pregnancy until 45 years. Clearly, strategy 1 with oocyte freezing at younger ages proved to be more efficient, with success rates of 84.5%, 52.3%, and 64.6%, respectively.
      Our return rate was nearly 10%, although they waited a mean of 2.2 years to return, which is still a short storage time. The reason may be related to collecting data from a relatively recent FP program. We can not forget that the purpose of this approach is to delay motherhood, and many women who decide to store their gametes are not yet willing to attempt pregnancy. In view of this, we expect a considerably increase in the return rate in the future. Recent data suggest that elective oocyte freezing is cost-effective in women under the age of 38 years (
      • Mesen T.B.
      • Mersereau J.E.
      • Kane J.B.
      • Steiner A.Z.
      Optimal timing for elective egg freezing.
      ,
      • Devine K.
      • Stillman R.J.
      • DeCherney A.H.
      Building a family through in vitro fertilization—economic realities.
      ). However, as shown in our study the highest success rates were achieved when patients were ≤35 years. Of course it is true that at the age 37–38 years, the chance of success might still be higher than in older patients (≥40 y) and that the return rate may be higher than for younger women, thus making it worthwhile to vitrify at these ages. Nevertheless, we should be very cautious with this interpretation, owing to the undeniable fact that from a biologic point of view the oocytes collected at younger ages provide the greatest chance of success, as we demonstrated here in an actual FP population. In our opinion, although earlier FP may be not as cost-effective as at 37–38 years of age, it is still advisable to counsel FP at younger ages owing to the higher probabilities of success. Another advantage is the higher chance of having not only one, but also a second child if the oocytes were stored earlier.
      A very frequent question that arises when discussing oocyte freezing for FP with patients is the number of oocytes required to maximize their chances of success in the future, which we could name the “number needed to freeze.” As with anything in medicine, individual variations here are so diverse because evaluating oocyte quality is still a conundrum. The best approach is chronologic age and chromosomal content, especially because we know that almost 80% of oocytes are already aneuploid at the age of 40 years (
      • Geraedts J.
      • Montag M.
      • Magli M.C.
      • Repping S.
      • Handyside A.
      • Staessen C.
      • et al.
      Polar body array CGH for prediction of the status of the corresponding oocyte. Part I: clinical results.
      ). As recently shown, one useful way to accurately assess success in IVF is to look at the cumulative probability of having a child according to the number oocytes consumed by means of the statistical approach with the use of Kaplan-Meier survival curves (
      • Garrido N.
      • Bellver J.
      • Remohi J.
      • Simon C.
      • Pellicer A.
      Cumulative live-birth rates per total number of embryos needed to reach newborn in consecutive in vitro fertilization (IVF) cycles: a new approach to measuring the likelihood of IVF success.
      ,
      • Cobo A.
      • Garrido N.
      • Pellicer A.
      • Remohi J.
      Six years' experience in ovum donation using vitrified oocytes: report of cumulative outcomes, impact of storage time, and development of a predictive model for oocyte survival rate.
      ). This approach offers a very precise analysis, because the probability of having a baby is provided at any moment according to the number of oocytes consumed, and is not derived by the absolute number achieved by the simple division of the number of babies by the number of oocytes. That approach is a much more simplistic, based on the false assumption that every oocyte has the same potential to become a child, which, as is well known, is not realistic. The analysis performed in our study enables us to picture the probability of having a baby, avoiding the calculation in absolute numbers. Obviously, the more oocytes the higher the probability, but the relationship is not linear, as shown by the curves, and is strongly related to a powerful confounder, i.e., the age of the patient. When we looked at our data, we observed a huge difference in CLBR when using only five oocytes (15.4%) compared with using eight (40.8%), which means an 8.4% increase in CLBR per additional oocyte if women were ≤35 years old. If they were ≥36 years old using the same number of oocytes, the increase in CLBR was considerably more modest (from CLBR of 5.1% with the use of five oocytes to 19.9% when eight oocytes were consumed, meaning an increase in CLBR of 4.9% per additional oocyte). Moreover, the success rate achieved in the younger group (≤35 y) was twice that achieved in the older group of women (≥36 y; 60.5% vs. 29.7%, respectively) when ten oocytes were used. With 15 oocytes, the CLBR continued to increase in the ≤35-year-old group, whereas with the same number of oocytes the plateau was already reached in the group of women aged ≥36 years, meaning that at this point the success is independent from the number of oocytes used up. In light of this, we suggest that at least eight to ten MII oocytes should be vitrified to obtain a reasonable success rate. In women older than 36 years, numbers should be individualized along with the possibility of offering PGS.
      In conclusion, we consider that our findings will contribute to confirm the huge impact that age has on fertility, to increase social awareness about the appropriate timing to consider and decide on FP, and to publicize the possibilities of this technology. Our data may well contribute to adequately counseling women who consult about FP for nonmedical reasons and to avoid unrealistic expectations drawn from general data. Women motivated by the desire to battle the age fertility decline should be encouraged to cryopreserve their eggs at a younger age to increase their future probabilities of having a biologic child. If there is a medical reason behind the decision, our data provide useful information about the possibilities of success according to the age of the patient.

      Acknowledgments

      The authors thank Mr. Goyo Iniesta for his valuable assistance in exploiting the data, Dr. Nicolás Garrido for his help with the Kaplan Meier analysis, and the entire staff of physicians, embryologists, and nurses in our group for enabling our fertility preservation program to operate.

      Appendix

      Figure thumbnail fx1
      Supplemental Figure 1Elective fertility preservation (FP) trends regarding the total number of oocyte vitrifications (vit.) for other reasons in our group. The plotting shows the total number of elective FP (EFP) cycles. All of the metaphase II (MII) vitrification cycles at all clinics involved. ∗∗Percentage of EFP cycles among the total MII vitrification cycles.
      Figure thumbnail fx2
      Supplemental Figure 2Distribution of elective fertility preservation (FP) cycles.
      Figure thumbnail fx3
      Supplemental Figure 3Marital status, sexual orientation, and level of education of women undergoing fertility preservation.
      Figure thumbnail fx4
      Supplemental Figure 4Distribution of ages at vitrification. (A) Elective fertility preservation (EFP) due to age. (B) EFP because of an associated medical condition.
      Figure thumbnail fx5
      Supplemental Figure 5Distribution of patients when returning according to age at vitrification and the mean storage time. (A) Elective fertility preservation (EFP) due to age. (B) EFP because of an associated medical condition.
      Supplemental Table 1Distribution of single and women with partner at vitrification and when returning, n (%)
      StatusEFP due to ageEFP due to medical reason
      Status at vitrification
       Single women95/120 (79.2)4/17 (23.5)
       Women with partner25/120 (20.8)13/17 (76.5)
      Status when returning
       Still single women
      Using donor sperm.
      50/120 (41.7)0
       Women with partner
      Using partners' sperm.
      70/120 (58.3)17 (100)
       Women single at vitrification and with partner when returning
      Using partners' sperm.
      45/95 (47.4)4/4 (100)
      Note: EFP = elective fertility preservation.
      a Using donor sperm.
      b Using partners' sperm.
      Supplemental Table 2Cryotransfers of surplus embryos.
      Parametern95% CI
      Patients with surplus embryos62NA
      Surplus embryos vitrified (per patient)185 (1.5 ± 2.1)0.97–20.3
      Patients with cryotransfers33NA
      Cryotransfers/patients (per patient)41/33 (1.2 ± 0.5)1.13–0.47
      Warming cycles43NA
      Embryos warmed (per warming cycle)98 (2.3)2.01–3.39
      Embryos transferred per cryotransfer61
      Three embryos were revitrified.
      /41 (1.5)
      1.22–1.58
      IR cryotransfer29.518.1–40.9
       CPR/warming cycle (%)17/43 (39.5)24.9–54.1
       OPR/warming cycle (%)11/43 (25.6)12.6–38.3
       CPR/transfer (%)17/41 (41.66)26.6–56.8
       OPR/transfer (%)11/41 (26.8)13.2–40.4
      Miscarriage rate/cycle (%)6/41 (14.6)3.8–25.4
      Live births9
      Four other pregnancies still ongoing.
      NA
      Note: CI = confidence interval; CPR = clinical pregnancy rate; IR = implantation rate; OPR = ongoing pregnancy rate.
      a Three embryos were revitrified.
      b Four other pregnancies still ongoing.
      Supplemental Table 3Clinical outcomes according to the reason for EFP.
      OutcomeEFP due to age95% CIEFP due to nononcologic medical reason95% CI
      No. of patients1,382NA86NA
      No. of cycles2,009NA128NA
      Mean age at vitrification, y37.737.5–37.935.7
      P<.05.
      34.9–36.3
      No. of retrieved oocytes (per patient)17,665 (12.7)12–12.21,250 (14.5)
      P<.05.
      12.9–13.7
      No. of retrieved oocytes (per cycle)17,665 (8.8)8.98–9.031,250 (9.7)8.9–9.5
      No. of MII oocytes vitrified (per patient)13,444 (9.7)9.6–9.9971 (11.2)
      P<.05.
      10.2–11.9
      No. of MII oocytes vitrified (per cycle)13,444 (6.7)6.6–6.9971 (7.6)6.4–7.9
      No. of patients returning120NA17NA
      Return rate (%)8.77.2–10.219.7
      P<.05.
      11.3–28.1
      Survival rate870/1,080 (80.5)78.1–82.9139/153 (90.9)
      P<.05.
      86.3–95.5
      Total no. of ETs/patient102NA15NA
      Total no. of embryos transferred154 (1.5)1.4–1.627 (1.8)1.5–2.1
      Implantation rate, %30.523.2–37.866.7
      P<.05.
      48.9–84.5
      CPR/transfer (%)42/102 (41.2)31.6–50.712/15 (80)
      P<.05.
      59.8–100
      CPR/patient (%)42/120 (35)25.7–44.312/17 (70.6)
      P<.05.
      48.9–92.3
      OPR/transfer (%)26/102 (25.5)17.0–33.911/15 (73.3)
      P<.05.
      50.9–95.7
      OPR/patient (%)26/120 (21.6)14.2–28.911/17 (64.7)
      P<.05.
      42.0–87.4
      No. of deliveries21NA5NA
      No. of live births24NA7NA
      Note: ET = embryo transfer; MII = metaphase II; other abbreviations as in Supplemental Table 1, Supplemental Table 2.
      a P<.05.

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