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Long-term cryopreservation of human oocytes does not increase embryonic aneuploidy

      Objective

      To determine if long-term cryopreservation of human oocytes affects oocyte developmental competence, blastocyst euploidy, or live-birth rates.

      Design

      Retrospective cohort study.

      Setting

      University-based fertility center.

      Patient(s)

      A total of 33 patients with cryopreserved oocytes underwent oocyte thaw, blastocyst culture, trophectoderm biopsy, and 24-chromosome preimplantation genetic screening (PGS) with array comparative genomic hybridization between December 2011 and July 2014; subjects were compared with 2:1 age-matched controls with fresh oocytes whose embryos underwent trophectoderm biopsy and PGS during the same period.

      Intervention(s)

      None.

      Main Outcome Measure(s)

      Rates of fertilization, blastulation, euploidy, implantation, and live birth.

      Result(s)

      Thirty-three patients (mean age 36.2 ± 3.8 y) thawed 475 oocytes that had been cryopreserved for a median of 3.5 years. Compared with 66 age-matched controls who underwent in vitro fertilization and PGS with fresh oocytes, embryos derived from cryopreserved oocytes demonstrated compromised blastocyst formation (54.5% vs. 66.2%) despite no impairment in fertilization (72.8% vs. 73.2%). Results showed no difference in the number of euploid blastocysts (1.7 ± 1.9 vs. 2 ± 2.5), percentage of euploid blastocysts (44.5% vs. 47.6%), rate of implantation (65% vs. 65%), or rate of live birth and ongoing pregnancy (62.5% vs. 55%) after 24-chromosome PGS with cryopreserved or fresh oocytes.

      Conclusion(s)

      Embryos derived from cryopreserved oocytes demonstrate impaired blastulation but equivalent rates of euploidy, implantation, and live birth compared with blastocysts derived from fresh oocytes, supporting the safety and efficacy of oocyte cryopreservation.

      Key Words

      Discuss: You can discuss this article with its authors and with other ASRM members at http://fertstertforum.com/goldmank-cryopreservation-oocytes-embryonic-aneuploidy/
      Oocyte cryopreservation (OC) is an effective means to preserve fertility in women at risk of medical- or age-related fertility loss (
      • Practice Committee of the American Society for Reproductive Medicine
      Mature oocyte cryopreservation: a guideline.
      ,
      • Stoop D.
      • van der Veen F.
      • Deneyer M.
      • Nekkebroeck J.
      • Tournaye H.
      Oocyte banking for anticipated gamete exhaustion (AGE) is a preventive intervention, neither social nor nonmedical.
      ,
      • Practice Committee of the American Society for Reproductive Medicine
      Fertility preservation in patients undergoing gonadotoxic therapy or gonadectomy: a committee opinion.
      ). The oocyte is particularly vulnerable to physical injury during the cryopreservation process, owing to its large volume and high water content, and cryopreservation causes alterations to the oocyte, including zona pellucida thickening and premature cortical granule exocytosis (
      • Ghetler Y.
      • Skutelsky E.
      • Ben Nun I.
      • Ben Dor L.
      • Amihai D.
      • Shalgi R.
      Human oocyte cryopreservation and the fate of cortical granules.
      ). Concerns have been raised regarding the risk of meiotic spindle disruption and aneuploidy in cryopreserved oocytes, and conflicting animal and human studies have demonstrated slow cooling– and vitrification-induced damage to the meiotic spindle (
      • Martinez-Burgos M.
      • Herrero L.
      • Megias D.
      • Salvanes R.
      • Montoya M.C.
      • Cobo A.C.
      • et al.
      Vitrification versus slow freezing of oocytes: effects on morphologic appearance, meiotic spindle configuration, and DNA damage.
      ,
      • Coticchio G.
      • Bromfield J.J.
      • Sciajno R.
      • Gambardella A.
      • Scaravelli G.
      • Borini A.
      • et al.
      Vitrification may increase the rate of chromosome misalignment in the metaphase II spindle of human mature oocytes.
      ,
      • Huang J.Y.
      • Chen H.Y.
      • Tan S.L.
      • Chian R.C.
      Effect of choline-supplemented sodium-depleted slow freezing versus vitrification on mouse oocyte meiotic spindles and chromosome abnormalities.
      ,
      • Kola I.
      • Kirby C.
      • Shaw J.
      • Davey A.
      • Trounson A.
      Vitrification of mouse oocytes results in aneuploid zygotes and malformed fetuses.
      ,
      • Tamura A.N.
      • Huang T.T.
      • Marikawa Y.
      Impact of vitrification on the meiotic spindle and components of the microtubule-organizing center in mouse mature oocytes.
      ,
      • Jimenez-Trigos E.
      • Naturil-Alfonso C.
      • Vicente J.S.
      • Marco-Jimenez F.
      Effects of cryopreservation on the meiotic spindle, cortical granule distribution and development of rabbit oocytes.
      ).
      Early clinical OC outcomes have been reassuring (
      • Noyes N.
      • Porcu E.
      • Borini A.
      Over 900 oocyte cryopreservation babies born with no apparent increase in congenital anomalies.
      ), and an eloquent sibling-oocyte study by Forman et al. demonstrated that oocytes vitrified for a brief time (15 min) were at no greater risk of aneuploidy or impaired implantation. However, by definition, women seeking OC intend on long-term gamete storage. To our knowledge, no data have been reported regarding the risk of aneuploidy and likelihood of live birth after prolonged oocyte cryo-storage. Outcome data after long-term OC are critical, particularly as the demand for and utilization of OC technology continues to grow.
      24-chromosome preimplantation genetic screening (PGS) has been shown to improve both neonatal and in vitro fertilization (IVF) outcomes (
      • Grifo J.A.
      • Hodes-Wertz B.
      • Lee H.L.
      • Amperloquio E.
      • Clarke-Williams M.
      • Adler A.
      Single thawed euploid embryo transfer improves IVF pregnancy, miscarriage, and multiple gestation outcomes and has similar implantation rates as egg donation.
      ,
      • Yang Z.
      • Liu J.
      • Collins G.S.
      • Salem S.A.
      • Liu X.
      • Lyle S.S.
      • et al.
      Selection of single blastocysts for fresh transfer via standard morphology assessment alone and with array CGH for good prognosis IVF patients: results from a randomized pilot study.
      ,
      • Forman E.J.
      • Hong K.H.
      • Ferry K.M.
      • Tao X.
      • Taylor D.
      • Levy B.
      • et al.
      In vitro fertilization with single euploid blastocyst transfer: a randomized controlled trial.
      ,
      • Forman E.J.
      • Hong K.H.
      • Franasiak J.M.
      • Scott Jr., R.T.
      Obstetrical and neonatal outcomes from the BEST Trial: single embryo transfer with aneuploidy screening improves outcomes after in vitro fertilization without compromising delivery rates.
      ), and in patients who have pursued 24-chromosome PGS using cryopreserved oocytes, PGS results can be used as an “assay” to assess the risk of meiotic spindle damage and aneuploidy after prolonged cryopreservation.
      We sought to compare outcomes of patients who pursued 24-chromosome PGS using cryopreserved and thawed oocytes (OC-PGS) with outcomes using fresh oocytes (IVF-PGS). The aims were 2-fold: (1) to determine if blastocysts from cryopreserved oocytes have a greater likelihood of aneuploidy than blastocysts from fresh oocytes; and (2) to compare pregnancy potential of blastocysts derived from cryopreserved vs. fresh oocytes following trophectoderm biopsy and PGS.

      Materials and methods

      A retrospective analysis was performed of OC-PGS and IVF-PGS cycles completed at the Fertility Center at New York University (NYU) Langone Medical Center. Approval was obtained from the Institutional Review Board of the NYU School of Medicine. Patients were included if they had undergone either slow-cooling or vitrification of oocytes between 2006 and 2014 and subsequently presented between December 2011 and July 2014 for oocyte thaw, blastocyst culture, and trophectoderm biopsy with array comparative genomic hybridization (aCGH) for 24-chromosome embryonic aneuploidy screening.
      In patients who underwent multiple oocyte retrieval procedures for OC, analysis was limited to oocytes obtained from the first procedure. All thaw-cycle data were analyzed for oocytes from the first retrieval procedure in order to appropriately compare outcomes with those in the control group of IVF patients for whom the biopsy results of all blastocysts are known. Five patients were included who had undergone polar body biopsy, oocyte vitrification, and subsequent thaw, blastocyst culture, trophectoderm biopsy, and rush day-6 embryo transfer (ET) under NYU Institutional Review Board Protocol 11-00395. All other ETs in this study were frozen ETs.
      Data on OC and PGS were compared with 2:1 age-matched controls, randomly selected using the Excel (Microsoft) random-number generator, who underwent a first cycle of IVF-PGS with trophoectoderm biopsy and aCGH with fresh oocytes during the same time period. Patients were excluded if they underwent PGS for a history of translocation or used donor oocytes. Only patients in each group with ≥1 blastocyst available for biopsy were included in order to compare rates of aneuploidy.
      Comparison parameters included: age at the time of oocyte retrieval; baseline serum follicle-stimulating hormone (FSH) and estradiol (E2) levels; total units of gonadotropin (International Units [IU]) used; peak serum E2 achieved on the day of ovulation trigger; total and number of mature (metaphase II [MII]) oocytes retrieved; 2-pronuclear (2PN) fertilization rate; blastocyst formation rate; total number of blastocysts biopsied; number of blastocysts biopsied on day 5, 6, and 7; and number of euploid and aneuploid embryos. The proportion of patients with no euploid embryos after biopsy was also reported.
      Data pertinent to OC cycles were abstracted, including the number of oocytes thawed after vitrification and slow-freezing, the total number of oocytes surviving thaw, and the percentage of oocytes surviving thaw. In patients who underwent ET, additional parameters analyzed included number of embryos transferred, implantation rate, clinical pregnancy rate, and live birth and ongoing pregnancy rate. The 2PN fertilization rate was expressed in terms of oocytes exposed to sperm, and “usable blastocyst” formation rate was defined as the number of good-quality blastocysts available for biopsy per 2PN fertilization. Implantation rate was defined as the number of gestational sacs per total number of embryos transferred, and clinical pregnancy rate was defined as the number of pregnancies with fetal cardiac activity per ET procedure. Only outcomes from the primary ET were included in the analysis.

       Ovarian Stimulation

      Before initiation of treatment, menstrual day 2 or 3 serum E2 and FSH levels were assessed. Patients with acceptable parameters (E2 <75 and FSH <13.5) underwent controlled ovarian hyperstimulation using injectable gonadotropins (follitropin [Merck Serono]; and menotropins [Ferring Pharmaceuticals]), with luteinizing-hormone (LH) suppression achieved using either a gonadotropin-releasing hormone agonist or antagonist. Ovulation was triggered when ≥2 follicles reached ≥17 mm in diameter, and ultrasound-guided transvaginal oocyte retrieval was performed 34–36 hours later.

       24-Chromosome Preimplantation Genetic Screening

      Laser-assisted breaching of the zona pellucida was performed on day 3 (Saturn, Research Instruments Ltd). Embryos were assessed on days 5, 6, and rarely 7, and fully differentiated good-quality blastocysts were biopsied. The trophectoderm cells extruding from the expanded blastocyst were gently pulled using suction, and a laser was used at cell junctions to remove cells without disrupting the inner cell mass. Biopsied trophectoderm cells were transferred into polymerase chain reaction tubes and sent to the reference laboratory for 24-chromosome analysis using aCGH as previously described (
      • Grifo J.A.
      • Hodes-Wertz B.
      • Lee H.L.
      • Amperloquio E.
      • Clarke-Williams M.
      • Adler A.
      Single thawed euploid embryo transfer improves IVF pregnancy, miscarriage, and multiple gestation outcomes and has similar implantation rates as egg donation.
      ,
      • Harton G.L.
      • Munne S.
      • Surrey M.
      • Grifo J.
      • Kaplan B.
      • McCulloh D.H.
      • et al.
      Diminished effect of maternal age on implantation after preimplantation genetic diagnosis with array comparative genomic hybridization.
      ). Following biopsy, blastocysts were vitrified to be replaced in subsequent frozen cycles, or in the case of a minority of patients, embryos underwent “rush” biopsy of a day-5 blastocyst followed by day-6 ET.

       Oocyte Cryopreservation and Thawing and/or Warming

      Oocyte cryopreservation and thawing and warming methods were performed according to those previously described by our group and are summarized below (
      • Grifo J.A.
      • Noyes N.
      Delivery rate using cryopreserved oocytes is comparable to conventional in vitro fertilization using fresh oocytes: potential fertility preservation for female cancer patients.
      ). Denuded oocytes noted to be metaphase II when evaluated 1.5 hours postharvest were considered suitable for cryopreservation. Both slow-freezing and vitrification methods were used, which was routine procedure at the time patients presented for OC.

       Slow-Freezing Method

      Oocytes were equilibrated for 10 minutes in a phosphate-buffered saline solution containing 1.5 mol/L propanediol (PROH), transferred into a phosphate-buffered saline loading solution containing 1.5 mol/L PROH plus 0.3 mol/L sucrose, and loaded into 0.25 ml cryopreservation straws (Conception technology, cat#006578). The temperature was reduced using a Planer Kryo 360 Controlled Rate Freezer (Planer Products Ltd.) with ice nucleation induced at –7°C. At –150°C, straws were transferred to liquid nitrogen tanks for storage. For warming, the straws containing the slow-cooled oocytes were air-warmed for 30 seconds, followed by brief placement in a 30°C water bath. Cryoprotectants were sequentially removed using stepwise dilution of the PROH and sucrose over a 30-minute period; surviving oocytes were transferred to fresh media for culture.

       Vitrification

      Vitrification was accomplished by placing oocytes in sequential equilibration solutions containing increasing concentrations of ethylene glycol and dimethyl sulfoxide, with the final equilibration solution containing 7.5% of both ethylene glycol and PROH. The gametes were then placed in the vitrification solution containing 15% ethylene glycol, 15% dimethyl sulfoxide, and 0.5 M of sucrose, for 90–110 seconds before being loaded into Cryotip (Irvine Scientific) or Cryolock (BioDiseño) containers. The devices were immediately sealed and capped and plunged into liquid nitrogen. With Cryotips, warming was achieved by agitating the containers in a 37°C water bath and releasing the straw contents into thaw media. For Cryolock containers, warming was achieved by rapidly submerging the tip containing the oocytes into prewarmed thaw solution. The recovered oocytes were passed sequentially through solutions containing decreasing concentrations of sucrose, to gradually remove the cryoprotectants. Finally, oocytes were washed in sucrose-free media and placed in culture media to allow them to recover before intracytoplasmic sperm injection (ICSI).

       Patient, Sperm Preparation, and Fertilization Method Used for Thaw and Transfer

      Patients scheduled for ET using previously cryopreserved oocytes underwent either day-5 rush biopsy and subsequent day-6 fresh transfer, or transfer of recryopreserved embryos from previously thawed oocytes. All patients undergoing ET underwent uterine preparation using sequentially increasing doses of oral ± transdermal ± vaginal E2 until the endometrial diameter reached ≥7 mm in greatest diameter; progesterone in oil (50 mg/d; Watson Pharmaceuticals) was added. Sperm preparation for all cycles involved either isolate (Irvine Scientific) or swim-up techniques as previously described. For OC cycles, ICSI was performed within 3 hours after oocyte thawing. Both ICSI and conventional insemination were used where appropriate for fresh IVF attempts.

       Statistical Analysis

      Univariate analysis was performed using the Student's t test with unequal variance or Fisher's exact test where appropriate. An alpha (α) error of <0.05 was considered statistically significant.

      Results

      Thirty-three patients cryopreserved 501 oocytes between June 2006 and February 2014 and subsequently presented for oocyte thawing, blastocyst culture, trophectoderm biopsy, and aCGH for PGS between December 2011 and July 2014. Patients cryopreserved oocytes for the following indications: deferred childbearing (n = 24), anticipated fertility loss due to medical conditions (n = 3), involvement in a study protocol (polar body biopsy study; n = 5), and unanticipated oocyte cryopreservation in lieu of IVF (n = 1). One patient who underwent oocyte thawing with the intention to pursue PGS had no blastocysts available for biopsy; she was therefore not included in the analysis.
      Oocytes were cryopreserved for a median of 3.5 years (range: 0–6.5 y). As was routine during the early part of the study period, both slow-freezing and vitrification were utilized. A total of 475 oocytes were thawed (vitrification: n = 322; 67.8%; slow-freezing: n = 153; 32.2%); survival rates after thawing or warming were 77.3% (n = 249) for vitrification, and 86.9% (n = 133) for slow-freezing. Sixty-six controls were matched to the mean OC patient age (36.2 ± 3.8 y). Cycle parameter comparisons between groups showed no statistically significant differences in baseline day-2 FSH or E2, total units of gonadotropin administered, E2 on the day of trigger, or total number of oocytes (total and metaphase II) retrieved (P>.05; Table 1).
      Table 1Patient and ovarian stimulation characteristics.
      VariableOC + PGS (n = 33)IVF + PGS (n = 66)P value
      Age at retrieval (y)36.2 ± 3.836.2 ± 3.81
      Age at trophectoderm biopsy (y)39.5 ± 5.336.2 ± 3.8.0005
      FSH (IU/mL)6.2 ± 26.3 ± 2.9.8
      E2 (pg/mL)42 ± 1845.5 ± 25.7.5
      Total gonadotropin (IU)3,340 ± 1,4303,740 ± 1,550.2
      E2 on day of trigger2,650 ± 1,4902,540 ± 1,120.7
      No. oocytes retrieved17 ± 8.615.2 ± 9.3.4
      No. metaphase II13.6 ± 6.312.8 ± 8.2.7
      Note: Data were analyzed with Student's t test and are presented as mean ± SD. IU = international units.
      On average, each OC patient thawed 14.4 oocytes, including 9.8 after vitrification and 4.6 following slow-freezing. A total of 80.4% of cryopreserved oocytes survived thawing. There was no difference in the number of 2PN zygotes (8.4 ± 5.8 vs. 9.4 ± 6.4, P=.5) or the rate of fertilization (72.8% vs. 73.2%, P=.9) using cryopreserved vs. fresh oocytes. The total number of blastocysts was lower in the OC group compared with the IVF group (4.6 ± 4 vs. 6 ± 5.1), but this number did not reach statistical significance (P=.1). The blastocyst formation rate was significantly lower in the OC-PGS group compared with the IVF-PGS group (54.5% vs. 66.2%, P<.001), as was the “usable blastocyst” rate (45.9% vs. 54.6%, P<.05) (Fig. 1).
      Figure thumbnail gr1
      Figure 1Outcomes following 24-chromosome preimplantation genetic screening with cryopreserved oocytes (OC + PGS) vs. fresh oocytes (IVF + PGS). *P<.05.
      No significant difference was seen in the mean number of blastocysts biopsied in the OC-PGS group compared with the IVF-PGS group (3.9 ± 3.6 vs. 5.1 ± 4.3, P=.2). There was a trend toward a lower percentage of day-5 blastocysts biopsied in the OC group (50.8% vs. 60.9%, P=.06), and a significantly greater percentage of day-6 blastocysts were biopsied in the OC group compared with controls (48.4% vs. 37.3%, P=.03). Notably, the mean number of euploid embryos was equivalent between the OC-PGS and the IVF-PGS groups (1.7 ± 1.9 vs. 2 ± 5, P=.6), and the percentage of euploid blastocysts was equivalent between groups (44.5% vs. 47.6%, P=.6). The percentage of patients with all aneuploid embryos after biopsy did not differ between groups (27.2% vs. 16.7%, P=.3) (Table 2).
      Table 2Laboratory outcomes of 24-chromosome preimplantation genetic screening with cryopreserved oocytes (OC + PGS) vs. fresh oocytes (IVF + PGS).
      VariableOC + PGS (n = 33)IVF + PGS (n = 66)P value
      Oocytes thawed14.4 ± 8.7
       Vitrification9.8 ± 8.6
       Slow-freezing4.6 ± 4.9
      Survival after thawing80.4
      Duration of cryo-storage (y), median3.5
      2PN8.4 ± 5.89.4 ± 6.4.5
      Fertilization rate72.873.2.9
      Total blastocysts4.6 ± 46 ± 5.1.1
      Blastocyst formation rate54.566.2.001
      Mean blastocysts biopsied3.9 ± 3.65.1 ± 4.3.2
      Day of biopsy
       Day 52 ± 2.93.1 ± 3.5.1
       Day 61.9 ± 1.72 ± 1.8.9
       Day 70.03 ± 0.20.1 ± 0.4.3
      Day of biopsy
       Day 550.860.9.06
       Day 648.437.3.03
       Day 70.81.7.7
      Usable blastocyst rate45.954.6.02
      Euploid embryos1.7 ± 1.92 ± 2.5.14
      Blastocyst euploidy44.547.6.6
      Patients with all aneuploid embryos27.216.7.3
      Note: Data are presented as mean ± SD (analyzed with Student's t test) or as % (analyzed with Fisher's exact test).
      Of the 33 patients in the OC-PGS group, and 66 age-matched controls, 48.5% (n = 16) of OC-PGS patients underwent ET, and 57% (n = 40) of controls underwent ET (P=.3). The mean number of embryos transferred was slightly higher in the OC-PGS group, as 100% of patients in the IVF-PGS group underwent single ET (mean number of embryos transferred, 1.3 ± 0.5 vs. 1 ± 0, P<.005). No differences were found in the implantation rate (65% vs. 65%, P=1), clinical pregnancy rate (62.5% vs. 57.5%, P=.8), or live birth and ongoing pregnancy rate (62.5% vs. 55%, P=.8). The numbers of supernumerary euploid embryos remaining were comparable in the two groups (1.3 ± 1.8 vs. 2 ± 2, P=.9) (Table 3). Outcomes were reanalyzed after excluding patients who underwent double ET; again, all outcomes were equivalent between groups. Pregnancy outcomes (clinical pregnancy rate and live birth–ongoing pregnancy rate) were additionally calculated using intention-to-treat analysis, with all patients in each group represented in the denominator. Again, the clinical pregnancy rate and live birth–ongoing pregnancy rate were similar between the OC and IVF groups (30.3% vs. 34.8%, P=.82).
      Table 3Frozen ET outcomes after 24-chromosome preimplantation genetic screening with cryopreserved oocytes (OC + PGS) vs. fresh oocytes (IVF + PGS).
      VariableOC + PGS (n = 33)IVF + PGS (n = 66)P value
      Underwent frozen ET48.5 (n = 16)60.6 (n = 40).3
      Embryos transferred1.3 ± 0.5 (n = 20)1 ± 0 (n = 40).005
      Implantation rate65651
      Clinical pregnancy rate62.557.5.8
      Live birth and ongoing pregnancy rate62.555.8
      Supernumerary euploid embryos1.3 ± 1.82 ± 2.9
      Note: Data are presented as mean ± SD (analyzed with Student's t test) or as % (analyzed with Fisher's exact test). Data were adjusted for number of embryos transferred; all outcomes remained nonsignificant.
      To account for any possible effect of the additional polar body biopsy intervention or rush ET on outcomes, data were reanalyzed after excluding those who underwent polar body biopsy and/or rush ET from the OC-PGS group (n = 6). Twenty-seven patients in the OC-PGS group were compared with the IVF-PGS group; no differences were seen in age, baseline FSH, units of gonadotropin, E2 (baseline and day of trigger), number of oocytes (total and metaphase II) retrieved, number of 2PN zygotes, or mean number of blastocysts biopsied. Again, a statistically significant impairment in blastocyst formation was seen in the OC group (53.5% vs. 66.2%, P<.001). The “usable blastocyst” rate was again lower in the OC-PGS group compared with controls (43.1% vs. 54.6%, P=.004). No differences were found in the number of euploid embryos, the percentage of euploidy, or the percentage of patients with all aneuploid embryos after biopsy.
      Because pregnancy outcomes differ in frozen ET cycles compared with fresh ET (
      • Shapiro B.S.
      • Daneshmand S.T.
      • Garner F.C.
      • Aguirre M.
      • Hudson C.
      Clinical rationale for cryopreservation of entire embryo cohorts in lieu of fresh transfer.
      ,
      • Shapiro B.S.
      • Daneshmand S.T.
      • Garner F.C.
      • Aguirre M.
      • Hudson C.
      • Thomas S.
      Evidence of impaired endometrial receptivity after ovarian stimulation for in vitro fertilization: a prospective randomized trial comparing fresh and frozen-thawed embryo transfer in normal responders.
      ), ET outcomes were reanalyzed after removing polar body biopsy and rush ET patients. Ten patients in the OC-PGS group underwent ET, compared with 40 in the control group. The majority of patients underwent single ET in both groups, but the number of embryos transferred was slightly higher in the OC-PGS group (1.2 ± 0.4 vs. 1 ± 0, P=.003). Comparisons between groups showed no differences in implantation rate (66.7% vs. 65%), clinical pregnancy rate (70% vs. 57.5%), or live birth and ongoing pregnancy rate (70% vs. 55%) (P>.05). The mean number of supernumerary euploid embryos cryopreserved was comparable between groups. Outcomes were reanalyzed after excluding patients who underwent double ET; all outcomes were equivalent between groups.

      Discussion

      Oocyte cryopreservation has revolutionized reproduction for women with cancer and, increasingly, for women seeking to defer reproduction (
      • Stoop D.
      • van der Veen F.
      • Deneyer M.
      • Nekkebroeck J.
      • Tournaye H.
      Oocyte banking for anticipated gamete exhaustion (AGE) is a preventive intervention, neither social nor nonmedical.
      ,
      • Noyes N.
      • Knopman J.M.
      • Melzer K.
      • Fino M.E.
      • Friedman B.
      • Westphal L.M.
      Oocyte cryopreservation as a fertility preservation measure for cancer patients.
      ,
      • Stoop D.
      • Cobo A.
      • Silber S.
      Fertility preservation for age-related fertility decline.
      ). Given the widespread adoption of OC technology and its marked projected growth, improving our understanding of the potential risks to the oocyte and developing embryo is imperative. Animal and human studies have demonstrated slow cooling– and vitrification-induced damage to the meiotic spindle, and data suggest that cryoprotectants may induce chromatin contraction and potentially jeopardize chromosomal competence (
      • Jimenez-Trigos E.
      • Naturil-Alfonso C.
      • Vicente J.S.
      • Marco-Jimenez F.
      Effects of cryopreservation on the meiotic spindle, cortical granule distribution and development of rabbit oocytes.
      ). However, studies suggest that metaphase spindles repolymerize, and chromatin properly realigns postcryopreservation, and we previously reported that 96% of oocytes demonstrating a visible spindle before freezing have one after thawing (
      • Noyes N.
      • Knopman J.
      • Labella P.
      • McCaffrey C.
      • Clark-Williams M.
      • Grifo J.
      Oocyte cryopreservation outcomes including pre-cryopreservation and post-thaw meiotic spindle evaluation following slow cooling and vitrification of human oocytes.
      ,
      • Smith G.D.
      • Motta E.E.
      • Serafini P.
      Theoretical and experimental basis of oocyte vitrification.
      ,
      • Gomes C.M.
      • Silva C.A.
      • Acevedo N.
      • Baracat E.
      • Serafini P.
      • Smith G.D.
      Influence of vitrification on mouse metaphase II oocyte spindle dynamics and chromatin alignment.
      ,
      • Bromfield J.J.
      • Coticchio G.
      • Hutt K.
      • Sciajno R.
      • Borini A.
      • Albertini D.F.
      Meiotic spindle dynamics in human oocytes following slow-cooling cryopreservation.
      ). Clinically significant damage to the meiotic spindle would be expected to result in a greater risk of aneuploidy; however, Forman et al. (
      • Forman E.J.
      • Li X.
      • Ferry K.M.
      • Scott K.
      • Treff N.R.
      • Scott Jr., R.T.
      Oocyte vitrification does not increase the risk of embryonic aneuploidy or diminish the implantation potential of blastocysts created after intracytoplasmic sperm injection: a novel, paired randomized controlled trial using DNA fingerprinting.
      ) recently demonstrated in a prospective study that oocyte vitrification did not increase the risk of aneuploidy in biopsied blastocysts.
      Pregnancy outcomes after OC have been reassuring; implantation rates after IVF with fresh and cryopreserved oocytes are comparable in a number of studies (
      • Forman E.J.
      • Li X.
      • Ferry K.M.
      • Scott K.
      • Treff N.R.
      • Scott Jr., R.T.
      Oocyte vitrification does not increase the risk of embryonic aneuploidy or diminish the implantation potential of blastocysts created after intracytoplasmic sperm injection: a novel, paired randomized controlled trial using DNA fingerprinting.
      ,
      • Goldman K.N.
      • Noyes N.L.
      • Knopman J.M.
      • McCaffrey C.
      • Grifo J.A.
      Oocyte efficiency: does live birth rate differ when analyzing cryopreserved and fresh oocytes on a per-oocyte basis?.
      ,
      • Siano L.
      • Engmann L.
      • Nulsen J.
      • Benadiva C.
      A prospective pilot study comparing fertilization and embryo development between fresh and vitrified sibling oocytes.
      ), as is the likelihood of live birth per oocyte (
      • Goldman K.N.
      • Noyes N.L.
      • Knopman J.M.
      • McCaffrey C.
      • Grifo J.A.
      Oocyte efficiency: does live birth rate differ when analyzing cryopreserved and fresh oocytes on a per-oocyte basis?.
      ). Postnatal outcomes of 900 babies born after OC demonstrated no increase in congenital anomalies compared with the general population (
      • Noyes N.
      • Porcu E.
      • Borini A.
      Over 900 oocyte cryopreservation babies born with no apparent increase in congenital anomalies.
      ).
      Mounting data support the safety of OC, the maintenance of DNA integrity after prolonged cryopreservation of other cell types, as well as the safety of long-term embryo cryopreservation (
      • da Cunha E.
      • Martins C.
      • Silva C.
      • Bessler H.
      • Bao S.
      Effects of prolonged in vitro culture and cryopreservation on viability, DNA fragmentation, chromosome stability and ultrastructure of bovine cells from amniotic fluid and umbilical cord.
      ,
      • Riggs R.
      • Mayer J.
      • Dowling-Lacey D.
      • Chi T.F.
      • Jones E.
      • Oehninger S.
      Does storage time influence postthaw survival and pregnancy outcome? An analysis of 11,768 cryopreserved human embryos.
      ). However, to our knowledge, no clinical data have been reported on the risk of embryonic aneuploidy after prolonged periods of OC. We sought to retrospectively compare the results of IVF with 24-chromosome PGS using cryopreserved and fresh oocytes to understand whether the OC process alters chromosomal competence.
      Our study population consisted of women who had cryopreserved oocytes with the intention of long-term cryo-storage, and subsequently returned, years later, for oocyte thawing when ready to pursue parenthood. This population represents the average OC patient seen in practice. Our results add to the growing body of OC safety literature by demonstrating no difference between cryopreserved and fresh oocytes in the number of euploid embryos after trophectoderm biopsy, the percentage of biopsied blastocysts that are euploid, or the number of supernumerary euploid embryos remaining after ET. These data further support the safety of OC, a crucial finding as use of this technology grows.
      In our study, blastocyst formation was significantly impaired after IVF with cryopreserved oocytes. This finding echoes existing literature suggesting that blastocyst formation is impaired after oocyte cryopreservation (
      • Forman E.J.
      • Li X.
      • Ferry K.M.
      • Scott K.
      • Treff N.R.
      • Scott Jr., R.T.
      Oocyte vitrification does not increase the risk of embryonic aneuploidy or diminish the implantation potential of blastocysts created after intracytoplasmic sperm injection: a novel, paired randomized controlled trial using DNA fingerprinting.
      ,
      • Goldman K.N.
      • Noyes N.L.
      • Knopman J.M.
      • McCaffrey C.
      • Grifo J.A.
      Oocyte efficiency: does live birth rate differ when analyzing cryopreserved and fresh oocytes on a per-oocyte basis?.
      ). The total blastocyst formation rate and the “usable” blastocyst formation rate were compromised, despite no impact on 2PN fertilization, suggesting that frozen-thawed oocytes are subject to impaired embryonic development after fertilization. These data suggest that meiotic spindle damage may in fact be occurring, but the abnormality may become apparent only during cell division as an “all-or-none” phenomenon in which incompetent (and possibly aneuploid) embryos arrest before the blastocyst stage. Therefore, if OC in fact contributes to aneuploidy, and “natural selection” in the laboratory selects against aneuploid blastocysts, this theory may explain the lower number of blastocysts available for biopsy or transfer.
      Thus, one limitation of our study is possible selection bias, as by definition, patients were included only if they had at least 1 blastocyst available for biopsy. However, only 1 patient was excluded from the OC group for lack of blastocysts for biopsy. Among the general IVF population intending to pursue PGS during this time period, 12%–15% of patients lacked blastocysts (or good-quality blastocysts) for biopsy.
      In addition to an impaired blastocyst formation rate, blastocysts derived from cryopreserved oocytes demonstrated developmental delay. A greater percentage of biopsied embryos were day-6 blastocysts in the OC group compared with the control group (48.4% vs. 37.3%, P<.05), supporting a possible insult to cell division from the cryopreservation process, leading to not only arrested growth but also delayed growth.
      Despite impaired blastocyst formation, pregnancy outcomes were comparable following IVF with 24-chromosome PGS using cryopreserved or fresh oocytes. All patients in the IVF-PGS control group underwent single ET, which itself represents a remarkable feat for patient safety. Four patients in the OC group underwent double ET; therefore, the number of embryos transferred was significantly greater in the OC group (1.3 ± 0.5 vs. 1 ± 0, P<.05). Although this difference was statistically significant, a difference of four patients is arguably not clinically significant. To account for this difference, outcomes were reanalyzed after excluding patients who underwent double ET, and pregnancy outcomes were again comparable between groups. Six patients in the oocyte cryopreservation group have not yet thawed all oocytes, and 26 oocytes remain cryopreserved. When accounting for these oocytes, reproductive potential could be even greater among the OC group. Although additional studies are needed, these data support the safety and efficacy of PGS using fresh or previously cryopreserved oocytes.
      The process of oocyte thawing, blastocyst culture, trophectoderm biopsy, and subsequent recryopreservation raises the question of the risk of “twice cryopreserving” gametes and embryos. Recent data suggest that twice-cryopreserved embryos have comparable live-birth potential compared with embryos cryopreserved just once, and that vitrified blastocysts can safely undergo thaw, trophectoderm biopsy, revitrification, and rethaw, with no detriment to outcomes (
      • Koch J.
      • Costello M.F.
      • Chapman M.G.
      • Kilani S.
      Twice-frozen embryos are no detriment to pregnancy success: a retrospective comparative study.
      ,
      • Taylor T.H.
      • Patrick J.L.
      • Gitlin S.A.
      • Michael Wilson J.
      • Crain J.L.
      • Griffin D.K.
      Outcomes of blastocysts biopsied and vitrified once versus those cryopreserved twice for euploid blastocyst transfer.
      ). Further studies are needed in this area to confirm the safety of twice-cryopreserving gametes.
      The limitations of our study are important to acknowledge. This study is retrospective in nature and would be best approached in a prospective manner. The sample size is relatively small, but given that OC and 24-chromosome PGS are relatively new technologies, we are likely among the first to publish results on the utilization of both concurrently. The 2:1 age-matched control group was randomly selected from all patients who pursued their first cycle of IVF-PGS during the same time that the OC patients underwent oocyte thawing and blastocyst biopsy, as a means to ensure consistency with biopsy stage and 24-chromosome screening platform. However, by definition, because oocytes had been cryopreserved for a median of 3.5 years in the OC group, the OC and IVF groups underwent oocyte retrieval during different time periods. Although little has changed in our facility regarding ovarian stimulation, the technical aspects of oocyte retrieval, or the conditions in the operating room or laboratory, we acknowledge that the difference in time period may introduce potential confounders. However, as age is undisputedly the most important prognostic factor, and currently the only universally accepted variable to predict embryo ploidy, patients were matched by “oocyte age” at the time of blastocyst biopsy.
      This study not only supports the safety of OC given comparable euploidy rates in blastocysts derived from cryopreserved and fresh oocytes, but also provides important information regarding the feasibility and efficacy of 24-chromosome PGS using cryopreserved oocytes. The PGS method has multiple proven benefits, including the advantage of enhanced embryo selection, leading to improved IVF outcomes and obstetric and neonatal outcomes (
      • Grifo J.A.
      • Hodes-Wertz B.
      • Lee H.L.
      • Amperloquio E.
      • Clarke-Williams M.
      • Adler A.
      Single thawed euploid embryo transfer improves IVF pregnancy, miscarriage, and multiple gestation outcomes and has similar implantation rates as egg donation.
      ,
      • Forman E.J.
      • Hong K.H.
      • Franasiak J.M.
      • Scott Jr., R.T.
      Obstetrical and neonatal outcomes from the BEST Trial: single embryo transfer with aneuploidy screening improves outcomes after in vitro fertilization without compromising delivery rates.
      ). In our practice, the utilization of 24-chromosome aneuploidy screening has increased significantly, from 28% of all IVF cycles in 2012 to nearly 50% of IVF cycles in 2014.
      The advantages of aneuploidy screening, and the concomitant increase in utilization, might additionally apply to patients with cryopreserved oocytes. In our study, the mean patient age in the OC-PGS group was 41 years (mean 40 years including polar body biopsy patients) at the time of blastocyst biopsy and ET, and the live birth and ongoing pregnancy rate in this group reached 70%. Selecting euploid blastocysts from autologous banked oocytes achieved live birth rates comparable to donor oocyte pregnancy rates, permitting a patient to effectively act as her own oocyte donor (
      • Knopman J.M.
      • Noyes N.
      • Grifo J.A.
      Cryopreserved oocytes can serve as the treatment for secondary infertility: a novel model for egg donation.
      ). The concomitant usage of PGS and OC technology may provide patients with the benefits of 24-chromosome PGS, namely an increased uptake of single ET, leading to improved obstetric and neonatal outcomes, while giving women the promise of reproductive autonomy after OC.
      Despite impaired blastocyst formation in embryos derived from cryopreserved oocytes, embryos that survive culture to the blastocyst stage after long-term OC display equivalent rates of euploidy, implantation, and live birth compared with blastocysts derived from fresh oocytes. The duration of oocyte cryo-storage (3.5 years) in our study reflects the reality of current practice, as women presenting for OC intend long-term gamete storage for fertility preservation. Our data suggest that long-term OC has no detrimental effect on chromosomal competence or live-birth outcomes, further supporting the safety and efficacy of this rapidly growing technology.

      Acknowledgments

      The authors thank Dr. David McCulloh and the physician, nursing, laboratory, and ancillary staff at the NYUFC, who all contributed to making the care of patients possible.

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