The present study reports, as far as is known for the first time, the safety of UV sterilization of liquid nitrogen and hermetical cryostorage of human oocytes by comparing the efficiency of fresh and vitrified sibling oocytes of infertile patients. A prospective randomized study on sibling oocytes of 31 patients was carried out. Metaphase-II oocytes were randomized for intracytoplasmic sperm injection and the supernumerary sibling oocytes were vitrified using a novel Cryotop aseptic procedure (UV liquid nitrogen sterilization and hermetical cryostorage). After unsuccessful attempts with fresh oocytes, vitrified sibling oocytes were injected. Mean outcome measures observed were fertilization, cleavage and top-quality embryo rates. No significant differences were observed between the fresh and vitrified-warmed sibling oocytes: oocyte fertilization was 88.3% versus 84.9%; cleavage 72.6% versus 71.0%; top-quality embryos 33.8% versus 26.3% and mean number of transferred embryos 2.6 ± 0.1 versus 2.5 ± 0.1, respectively. Clinical pregnancy rate per cycle with vitrified-warmed oocytes was 35.5% (implantation rate 17.1%) and seven healthy babies were born. This study demonstrated that UV liquid nitrogen sterilization and hermetical cryostorage does not adversely affect the developmental competence of vitrified oocytes, allowing safe aseptic open vitrification applicable under strict directives on tissue manipulation.

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Nowadays, human oocytes/embryos are cryopreserved via slow freezing or vitrification. The aim of this study was to evaluate a rapid warming protocol for slow-frozen human oocytes based on the standard warming procedure for vitrification. This was a prospective study on 216 sibling oocytes randomized for either conventional rapid thawing or rapid warming with vitrification warming solution. The primary endpoint was morphological assessment of survival at 2h. Surviving oocytes were divided into two subgroups: (i) parthenogenetically activated; and (ii) fixed and observed for spindle/chromosome configuration. Secondary endpoints were parthenogenetic development and spindle/metaphase configuration. Survival rate with rapid warming was higher (92/102, 90.2%) than with rapid thawing (85/114, 74.6%; P=0.005), and after 3d of culture the rapidly warmed parthenotes had more blastomeres compared with those rapidly thawed (P=0.042). Meiotic spindle and chromosomal configuration were not significantly influenced by rapid warming or rapid thawing. The finding of this study allows IVF centres to increase the efficiency of oocyte slow freezing, enabling survival rates comparable to vitrification protocols, and potentially to optimize costs by using the same warming protocol for both slow-frozen and vitrified reproductive cells.

PurposeTo study the efficacy and efficiency of a “universal warming protocol” for vitrified human embryos, based on subsequent steps with 1 and 0.5 M concentration of extracellular cryoprotectant (ECCP).MethodTwo studies on patients undergoing fertility treatments via ICSI: a prospective randomized controlled trial (RCT) and a retrospective cohort study (CS). Setting: Private assisted reproductive (AR) center.RCT: duration 01/03/2017–01/10/2017; 315 embryos at blastocyst stage obtained from 169 patients. Each patient’s embryos were first randomized for vitrification with two different kits: Vitrification Kit (Kitazato, Japan) and Sage Vitrification Kit (Origio, Denmark). The embryos were randomly warmed with either Kitazato (K) or Sage (S) warming kits, specifically: group A (KK), group B (KS), group C (SK), and group D (SS). Primary outcome measure: survival rate (number of embryos surviving per number of embryos warmed). Secondary: implantation rate (number of embryos implanted per number of embryos transferred).CS: duration 01/01/2013–31/12/2015 embryos from patients’ own oocytes; 10/04/2015–31/07/2017 embryos from donors’ oocytes. A total of 1055 embryos vitrified at cleavage stage obtained from 631 warming cycles: 847 of these obtained from patients’ own oocytes, 208 egg-donation-derived embryos. Each patient’s embryos were vitrified and warmed in various combinations of three different vitrification/warming kits: Kitazato (K), Sage (S), or made in-house in our laboratory (H). Vitrification/warming kits from different manufacturers are routinely used in our AR center, and the warming procedures are randomly performed with any available kit on a “first-in-first-out” basis, irrespective of the kit used for vitrification. Group names: KK, KS, SK, SS, SH, HK, HS, HH (embryos from patients’ own oocytes); eKK, eKS, eSK, eSS (egg-donation-derived embryos).ResultsCryo-survival rates were comparable in all study groups.RCT. Group A 99.0% (96/97), group B 98.8% (83/84), group C 98.4% (61/62), and group D 98.6% (71/72).CS. Embryos from patients’ own oocytes: KK 96.4% (54/56), KS 100.0% (13/13), SK 98.8% (80/81), SS 97.2% (174/179), SH 97.6% (40/41), HK 95.2% (20/21), HS 99.5% (187/188), and HH 97.4% (261/268). Egg-donation-derived embryos: eKK 100.0% (91/91), eKS 98.4% (60/61), eSK 100.0% (26/26), and eSS 96.7 (29/30).Implantation was generally comparable in all study groups—exceptions were in CS: KS vs. SK (P = 0.049), SS (P = 0.012), HS (P = 0.010), HH (P = 0.025); and SH vs. SS (P = 0.042), HS (P = 0.035).ConclusionWorldwide, millions of embryos have been cryopreserved using different vitrification kits; these studies establish that it is possible to combine different kits for vitrification and warming using a universal warming protocol. This can optimize costs, simplify lab routines, and favor embryo exchange between IVF centers.RCT registration numberISRCTN12342851.

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vitrification (CV) in a paired design. Cryopreserved tissues were thawed 8-10 weeks later and evaluated on thaw (0h) and after 24-96h culture. Formalin fixed, paraffinembedded blocks were serially sectioned and primordial (pdf) and primary (pyf) follicle densities, % of pdf and pyf with DNA double-strand breaks (by gH2AX) and apoptotic cell death pathway activation (AC3) were determined. RESULTS: The data are summarized in Table 1. Compared to OV, pdf and pyf densities were similar in the CV group after thawing. Likewise, the percentages of apoptotic and DNA-damaged pdf did not differ between the OVand CV groups on the thaw day. However, the percentage of apoptotic pdf was significantly reduced in the CV group compared to the OV after 96h in culture (p¼0.03). CONCLUSIONS: Our results show that the newly developed CV system provides similar or better efficacy compared to open vitrification with human ovarian tissue. The mechanism of higher primordial follicle survival rate with CVafter 96h culture needs to be further explored. Having showed that the OV has similar efficacy to slow freezing with ovarian tissues (see abstract 2017-A-2005-ASRM), we propose that the CV method should become the standard method of ovarian tissue cryopreservation.

for confounders, logistic and Poisson regression models, adjusted a priori for oocyte age, donor egg, and serum P measurement on day 3, were used to estimate the odds ratio (OR) or relative risk (RR) and 95% confidence interval (CI) of live birth. RESULTS: Mean serum P at transfer on day 5 was 28.4AE10.5 ng/mL. Compared to patients with P>20 ng/mL, patients with P<20 ng/mL on day 3 or 5 had a significantly higher implantation failure rate (negative hCG, 30.8% vs 22.3%, OR 1.54, 95% CI 1.07-2.22), lower CPR (57.9% vs 67.8%, OR 0.66, 95% CI 0.47-0.91) and lower LBR (45.1 vs 58.1%, OR 0.60, 95% CI 0.43-0.83), even after increasing P dose (Table). LBRs were stable from serum P 20 to 60 ng/mL (assay upper limit); there was no upper limit of P above which the LBR declined. Among obese patients (BMI>30 kg/m2, n¼129), P<20 ng/mL was more common than in non-obese patients (n¼672) (52.7% vs 18.9%, OR 4.76, 95% CI 3.2-7.1). Among patients with P>20 ng/mL, the LBR was significantly lower in obese vs. non-obese patients (44.6 vs 57.7%, OR 0.60, 95% CI 0.38-0.96). CONCLUSIONS: Serum P>20 ng/mL was associated with increased CPR and LBR following blastocyst transfer into a prepared uterus. LBRs were not rescued by increasing P doses for serum levels <20 ng/mL. Obese recipients may require higher initial doses of IM P for luteal support. Future research is needed to define the optimal serum P at transfer, and to determine whether this varies according to patient characteristics, such as BMI.