The kitazato “closed” cryotop sc vitrification system performs comparably to its original “open” system. a study using unfertilized human eggs, mouse eggs, and mouse embryos

The kitazato “closed” cryotop sc vitrification system performs comparably to its original “open” system. a study using unfertilized human eggs, mouse eggs, and mouse embryos

MATERIALS AND METHODS: We retrospectively reviewed in vitro fertilization cycles in 264 320 patients undergoing in vitro fertilization (IVF) at an aca...

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MATERIALS AND METHODS: We retrospectively reviewed in vitro fertilization cycles in 264 320 patients undergoing in vitro fertilization (IVF) at an academic fertility center. We included all all autologous FOET cycles (n¼57) and all frozen embryo transferFET cycles (n¼272) between 2012-2014. Fresh IVF, oocyte donor cycles and blastocyst transfers were excluded. Patient demographics and cycle characteristics were abstracted from chartscollected. Outcomes were compared between FOET and FET groups using T-test, chi-square analysis, and multivariable logistic and linear regression to adjust for age at the time of cryopreservation. RESULT(S): 264 patients and 264 patients and 329 cycles were included in our analysis. Patients in the FOET group were younger at the time of their oocyte oocyte retrieval compared to FET patients (33.1 vs 36.4 years, p< .00132.8 vs 36.4 years, p<0.001). There were no significant differences in demographics, ovarian reserve markers, or cycle stimulation parameters. Mean survival of thawed oocytes was lower than that of thawed embryos (76.8+2.23 versus 94.3+0.76, p < .001), even when stratified by cryopreservation method (p < .001). When controlling for age, regression analysis showed no differences in implantation, clinical pregnancy rate (CPR), and live birth rates (LBR), or obstetrical outcomes between the FOET and FET groups (Table 1). When the analysis was limited to women > 38, there were no differences in clinical pregnancyCPR or live birth ratesLBR between FOET and FET groups (Table 2). In the FOET group, the oldest age at the time of cryopreservation to result in a live birth was 41 years old. CONCLUSION(S): OC achieves live birth rates that are comparable to embryo cryopreservation, and is a viable option even for women of advanced reproductive ages. Our findings suggest that women considering fertility preservation can be counseled that OC is as effective as embryo cryopreservation. SUPPORT: None. References:

matched donors and a large reference infertility population which excluded donors. RESULTS: 169 women met inclusion criteria. Average age of donors was 26 (range 22-31), and average number of blastocysts analyzed was 8.7 (range 1-32). The rate of aneuploidy overall is significantly higher in the general IVF population compared to the donor population, 26.6% compared to 19.2% (p<0.0001). When looking specifically at women younger than 26, the rate of aneuploidy in the general IVF population is 34% compared with 18.5% in the donor population (p<0.0001). Above age 27, there is no clear difference seen (Figure 1). CONCLUSION: The rate of aneuploidy is higher in the infertile IVF population when compared to the donor population at young ages. However, it is notable that the prevalence of aneuploidy in the donor population was still approximately 20%. While donor oocyte does provide a lower rate of aneuploidy than in the infertile population, the risk is not completely avoided and preimplantation genetic screening still provides a meaningful method of embryo selection. FINANCIAL SUPPORT: None. Reference: 1. Franasiak, Jason M., et al. The nature of aneuploidy with increasing age of the female partner: a review of 15,169 consecutive trophectoderm biopsies evaluated with comprehensive chromosomal screening. Fertility and Sterility. Volume 101, Issue 3: 656-663.e1.

TABLE 1. Outcomes For Women > 38 At The Time of Cryopreservation

FOET (n¼14)

FET (n¼116)

P-value

8.1% 3 (21.4%) 3 (21.4%)

13.1% 24 (20.7%) 21 (18.1%)

0.25 0.27 0.3

b

Implantation Rate Clinical Pregnanciesa Live Birthsa a

Values depicted as mean (percentage). Calculated by total # gestational sacs/ total # embryos transferred.

b

Figure 1. Aneuploidy rates of screened embryos from a donor oocyte population compared to a reference infertile population.

P-28 P-27 ANEUPLOIDY RATE IN DONOR OOCYTE CYCLES REMAIN SUBSTANTIAL, BUT ARE LOWER WHEN COMPARED TO GENERAL INFERTILITY POPULATION. J. Horne, J. Franasiak, M. Olcha, C. R. Juneau, S. J. Morin, P. A. Bergh, R. T. Scott. Rutgers, Robert Wood Johnson; Reproductive Medicine Associates of New Jersey.

THE KITAZATO ‘‘CLOSED’’ CRYOTOP SC VITRIFICATION SYSTEM PERFORMS COMPARABLY TO ITS ORIGINAL ‘‘OPEN’’ SYSTEM. A STUDY USING UNFERTILIZED HUMAN EGGS, MOUSE EGGS, AND MOUSE EMBRYOS. T. T. F. Huang.a,b aUniversity of Hawaii John A. Burns School of Medicine, Department of Obstetrics and Gynecology, Honolulu, HI; bPacific In Vitro Fertilization Institute, Kapiolani Medical Center, Honolulu, HI 96826.

BACKGROUND: Aneuploidy is a major contributor to decreased reproductive potential and is more prevalent with increasing age. Additionally, there is a surprisingly high occurrence of aneuploidy in infertile patients at very young ages (1). Donor oocytes are commonly used as a therapeutic option for older women. However, observed aneuploidy in very young IVF patients suggests young patients may also be prone to higher rates of aneuploidy. A large comparison of aneuploidy rates in infertile patients and oocyte donors utilizing a 24 chromosome aneuploidy platform has not been done. OBJECTIVE: To determine the rate of aneuploidy in the donor population compared with the general IVF population. MATERIALS AND METHODS: Patients from a single institution who underwent donor oocyte cycles with comprehensive chromosome screening (CCS) from 2009 through 2014 were included. Oocyte donors underwent routine ovarian stimulation and oocyte retrieval. After extended culture and trophectoderm biopsy, CCS was performed utilizing a validated platform. Chi-squared was utilized to compare aneuploidy rates between age

BACKGROUND: Vitrification has become the preferred cryopreservation method for human eggs and embryos (Vajta, 2015). Most approaches utilize direct ‘‘open’’ exposure to liquid nitrogen (LN2) to achieve maximal cooling rates. However, closed systems are safer from theoretically toxic effects from exposure to LN2 (Bielanski and Vajta , 2009; Vajta 2015). A new ‘‘closed’’ vitrification system is the ‘‘Cryotop SC’’ (Kitazato Corp., Shizuoka, Japan). Cryotops are cooled within a plastic containment straw  without direct LN2 contact. The cooling rate is approximately -2000 C /min compared to > -70,000 C/min. OBJECTIVE: This study is a head-to-head trial of Kitazato’s open and closed systems using unfertilized eggs (mouse and human) and 8-cell and blastocyst stage mouse embryos. MATERIALS AND METHODS: Metaphase II human eggs (n¼50) from egg donation cycles were obtained from Metaphase I eggs matured to Met II. Fresh unfertilized Metaphase II mouse eggs (n¼56) were provided courtesy of the Institute for Biogenesis Research, University of Hawaii.

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PCRS Abstracts

Vol. 105, No. 2, Supplement, February 2016

Two-cell stage mouse embryos were thawed from a commercial source (Embryotech, Haverhill, MA). Embryos were cultured in Global Total and vitrified at either the 8-cell (n¼50) or early blastocyst stages (n¼50). VITRIFICATION: Material was divided equally between each protocol. Cells were treated in ES solution for 6-8 minutes, followed by VS solution for 60 seconds before plunging directly into LN2 (open system) or placing into a supercooled containment straw (closed system). WARMING: For the ‘‘closed’’ system, the straw was cut while in LN2; the cryotop was then moved to 37 0C TS. Subsequent steps were identical for each group. Statistical comparisons used Chi-square. RESULTS: Figure 1 shows survival of Met II human and mouse eggs after culture overnight. No differences were seen in either recovery or survival between open or closed systems (p >0.5). Notably, all human eggs in the experiments were successfully recovered using the closed SC system. Figure 2 shows the recovery, survival, and development of 8-cell mouse embryos to the blastocyst stage. No differences were seen in survival (p >0.4) or expansion (p> 0.2) to hatching over 24-36 hours. Furthermore, there were no differences in survival or expansion of vitrified blastocysts (p > 0.3). CONCLUSIONS: The new ‘‘closed’’ Kitazato SC vitrification system produces comparable recovery, survival and developmental rates as the traditional ‘‘open’’ system when using human eggs, mouse eggs and mouse embryos. Results support the importance of warming rates with slower cooling rates (Mazur and Seki, 2011). SUPPORT AND ACKNOWLEDGEMENTS: This work was supported by the Pacific IVF Institute and the Department of Obstetrics and Gynecology of the University of Hawaii John A. Burns School of Medicine. We also thank the Kitazato Corporation for kindly providing all vitrification supplies and reagents. We also thank the Institute for Biogenesis Research at the University of Hawaii for generously provided fresh mouse eggs. There is no financial relationship between Kitazato Corporation with the author or the University of Hawaii. Work proceeded in accordance with IRB approved protocols for human unfertilized eggs and mouse embryo. References: 1. Bielanski A, Vajta G. 2009. ‘‘Risk of contamination of germplasm during cryopreservation and cryobanking in IVF units.’’ Hum Reprod. 24:2457-67.

Figure 1. Human (blue) mouse (red) Met II eggs.

Figure 2. 8-cell development to blastocyst.

FERTILITY & STERILITYÒ

Figure 3. Expansion of vitrified blastocysts.

2. Mazur P, Seki S. 2011. ‘‘Survival of mouse oocytes after being cooled in a vitrification solution to -196 degrees C at 95 degrees to 70,000 degrees C/min and warmed at 610 degrees to 118,000 degrees C/min: A new paradigm for cryopreservation by vitrification,’’ Cryobiology 62:1-7. 3. Vajta G, Rienzi L, Ubaldi FM. 2015. ‘‘Open versus closed systems for vitrification of human oocytes and embryos,’’ Reprod Biomed Online 30:325-333.

P-29 EFFECTS OF CYCLOPHOSPHAMIDE AND MESNA ON METAPHASE II MOUSE OOCYTE QUALITY. Roohi Jeelani,a Faten Shaeib,a Mili Thakur,a,b Sana Khan,a Husam M. Abu-Soud.a aDepartment of Obstetrics and Gynecology, The C.S. Mott Center for Human Growth and Development, Wayne State University School of Medicine, Detroit, MI, 48201; bDivision of Genetic and Metabolic Disorders, Department of Pediatrics and Center for Molecular Medicine and Genetics, Wayne State University, Detroit, MI, 48201. BACKGROUND: Cyclophosphamide (CY) is a well known chemotherapeutic agent used to treat ovarian, breast, and hematological cancers and autoimmune disorders. Acrolein (a highly electrophilic, a, b-unsaturated aldehyde) and phosphoramide mustard are the two major cytotoxic metabolites of CY. In addition, humans may also be exposed to acrolein through a variety of other sources including cigarette, industrial, and environmental exposure. Phosphoramide mustard works by introducing alkyl radicals into biologically active molecules inducing cell apoptosis. Mesna, used as an adjuvant during chemotherapy, is an organosulfur given to patients in order to reduce the undesired side effects of CY. Females exposed to CY based chemotherapy experience ovarian failure however the exact mechanism remains unknown. OBJECTIVE: We hypothesized that CY and its major metabolites affect female fertility through a mechanism that involves the deterioration of oocyte quality. Material and Methods: In our current work, we investigated the effects of cyclophosphamide, acrolein, phosphoramide mustard, as well as mesna on metaphase II mouse oocytes (n¼150) through exposure to increasing concentrations of each molecule (0, 5, 10, 20, 50, and 100 mM) for 4 hours. Oocytes were fixed and subjected to indirect immunofluorescence for detecting changes in microtubule morphology (MT) and chromosomal alignment (CH) using a previously validated 1-4 scoring system. RESULTS: Collectively, our results show treatment of oocytes with increasing concentrations of CY shows a linear increase in poor scoring for both spindle structure and CH alignment, which plateaued at 50 mM. In contrast, acrolein showed more damaging effects as judged by the shift of the curve to the left. Phosphoramide mustard showed no significant or slight increase in poor scoring of both CH and MT upon increasing the drug concentration. Importantly, exposure to mesna also adversely affected the oocyte quality, similar to the deterioration seen with CY. CONCLUSION: All treatment groups showed poor scoring even when treated with the chemo protective drug, mesna. Deterioration in oocyte quality in all treatment groups was mediated through the production of ROS. Therefore, role of therapeutic options like antioxidant supplements should be investigated to counteract the effect of CY based chemotherapy and preserve fertility in women undergoing such treatment. FINANCIAL SUPPORT: None.

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