Vitrification in Open and Closed Carriers at Different Cell Stages: Assessment of Embryo Survival, Development, DNA Integrity and Stability during Vapor Phase Storage for Transport

<p>Abstract</p> <p>Background</p> <p>High cooling rates with vitrification can be achieved through the use of carriers that allow cryopreservation in fluid volumes < one μl. Open carriers allow direct contact of embryos with liquid nitrogen (LN2) whereas closed carrier systems sequester the embryo within a sealed system during immersion in LN2. The use of closed systems may be preferable to reduce the possibility of cross-contamination. In the present study, we compare open and closed carriers for vitrification of embryos. We also examine their ability to retain embryo viability during vapor phase transport.</p> <p>Methods</p> <p>Frozen one-cell mouse embryos were thawed and randomly allocated to treatment groups. Embryos were cultured and vitrified at the 8-cell (CL) or at the blastocyst (BL) stage. The cryoloop, an open carrier was tested against two closed systems, the Cryotip and the HSV straw. Carriers were tested for their ability to maintain embryo viability when held in the vapor phase of a dry shipper for a period of 96 hours. Outcome parameters monitored were embryo survival, recovery, subsequent development and signs of DNA damage.</p> <p>Results</p> <p>A total of 561 embryos were vitrified. The only parameter significantly affected by the type of carrier was the percentage of embryos recovered after warming. Vitrification of both CL and BL stage embryos in the Cryotip resulted in significantly lower recovery rates (P < 0.001). The subsequent developmental parameters were unaffected by either the carrier or the cell stage. Vapor phase storage for 96 hours under "transport conditions" did not appear to adversely affect the viability after warming. Quantitative analysis for DNA damage showed that <5% of cells were TUNEL positive. Interestingly, the overall percent of cells exhibiting DNA damage was lower after CL stage vitrification (P < 0.001).</p> <p>Conclusion</p> <p>This study is one of the first to examine DNA integrity after vitrification on different carriers and at different cell stages. It also provides insight on relative safety of short term vapor storage of vitrified embryos during transport. Within the limits of this study we could not detect an adverse effect of vapor storage on blastomere DNA or other measured outcome parameters.</p

Severe teratozoospermia and its influence on pronuclear morphology, embryonic cleavage and compaction

<p>Abstract</p> <p>Background</p> <p>Fertilization, cell division and embryo development depend on genomic contributions from male and female gametes. We hypothesize that teratozoospermic sperm influences early embryo development and embryo compaction.</p> <p>Methods</p> <p>We conducted a retrospective analysis of embryos derived from intracytoplasmic sperm injection (ICSI) cycles. Two hundred thirty-five consecutive ICSI cycles were included in the study; all treatment was provided at the Cleveland Clinic Fertility Center. Patient cycles were divided by sperm morphology based on Kruger's strict criteria: Group A, embryos where teratozoospermic sperm (0-2% normal) were used for ICSI and Group B, embryos where dysmorphic sperm (5-13% normal) were used for ICSI. All cycles analyzed were of patients doing day 3 embryo transfers. Outcome measures assessed included pronuclear (PN) pattern, syngamy, early cleavage, cell number, rate of compaction and blastulation of embryos left in culture and not transferred on day 3.</p> <p>Results</p> <p>A total of 1762 embryos were analyzed. PN patterns were similar in Group A and Group B embryos. No differences were noted in syngamy, cleavage, cell number or blastulation rate. Studying the development of embryos in culture after day 3 transfer revealed a difference in the timeline for compaction. By day 4, 25% of Group A embryos had compacted compared to 36% in Group B (P = 0.0007). There was no difference found between Group A and Group B embryos in regards to blastulation.</p> <p>Conclusions</p> <p>We did not find an association between sperm morphology and clinical outcomes. The impact of teratozoospermia may be masked in ICSI cycles where fertilization, implantation rate and clinical pregnancy rate are the primary outcome measures. However, by examining the timeline of development, we were better able to discern a potential paternal effect at critical transition points from fertilization through activation.</p

Three dimensional culture of fresh and vitrified mouse pre-antral follicles in a hyaluronan-based hydrogel: a preliminary investigation of a novel biomaterial for in vitro follicle maturation

Abstract Background Folliculogenesis within the ovary requires interaction between somatic cell components and the oocyte. Maintenance of 3-dimensional (3-D) architecture and granulosa-oocyte interaction may be critical for successful in vitro maturation of follicles. Testing of novel biomaterials for the 3-D culture of follicles may ultimately lead to a culture model that can support the longer in vitro culture intervals needed for in vitro maturation of human oocytes from ovarian tissue biopsies. Methods A novel tyramine-based hyaluronan (HA) hydrogel was tested for its biocompatibility with ovarian follicles. The HA was prepared at concentrations from 2 to 5 mg/ml. HA hydrogel was also formulated and tested with matrix proteins (ECM). Enzymatically isolated pre-antral follicles from the ovaries of 10–12 day SJL pups were divided amongst control (CT) and HA treatments. The growth of both fresh and vitrified follicles was assessed after encapsulation in the hydrogel. The basal culture medium was MEM alpha supplemented with FSH, LH, ITS and 5% FBS. Maturation was triggered by addition of hCG and EGF after in vitro culture (IVC). Outcome parameters monitored were follicle morphology, survival after IVC, antrum formation, GVBD and MII formation. Differences between treatments were analyzed. Results HA and ECM-HA encapsulated follicles looked healthy and maintained their 3-D architecture during IVC. In control cultures, the follicles flattened and granulosa:oocyte connections appeared fragile. Estradiol secretion per follicle was significantly higher by Day 12 in ECM-HA compared to HA or CT (4119, 703 and 1080 pg/ml, respectively). HA and ECM-HA cultured follicles had similar survival rates (62% and 54%, respectively), percent GV breakdown (96–97%), MII formation (47–48%) and oocyte diameters at the end of IVC. Control cultures differed significantly in percent GVBD (85%) and MII formation (67%) . Vitrified-warmed follicles encapsulated in HA had an oocyte maturation rate to MII of 54% as compared to 57% in non-embedded follicles. Conclusions Initial testing of this new and unique HA-based hydrogel was quite promising. The ease of follicle encapsulation in HA, its optical transparency and ability to be molded combined with its support of follicle growth, estradiol secretion and resumption of meiosis make this HA-hydrogel particularly attractive as model for 3-D ovarian follicle culture.</p

Three-dimensional in vitro follicle growth: overview of culture models, biomaterials, design parameters and future directions

In vitro ovarian follicle culture is a new frontier in assisted reproductive technology with tremendous potential, especially for fertility preservation. Folliculogenesis within the ovary is a complex process requiring interaction between somatic cell components and the oocyte. Conventional two-dimensional culture on tissue culture substrata impedes spherical growth and preservation of the spatial arrangements between oocyte and surrounding granulosa cells. Granulosa cell attachment and migration can leave the oocyte naked and unable to complete the maturation process. Recognition of the importance of spatial arrangements between cells has spurred research in to three-dimensional culture system. Such systems may be vital when dealing with human primordial follicles that may require as long as three months in culture. In the present work we review pertinent aspects of in vitro follicle maturation, with an emphasis on tissue-engineering solutions for maintaining the follicular unit during the culture interval. We focus primarily on presenting the various 3-dimensional culture systems that have been applied for in vitro maturation of follicle:oocyte complexes. We also try to present an overview of outcomes with various biomaterials and animal models and also the limitations of the existing systems

Paternal effect on genomic activation, clinical pregnancy and live birth rate after ICSI with cryopreserved epididymal versus testicular spermatozoa

<p>Abstract</p> <p>Background</p> <p>This study takes an in depth look at embryonic development, implantation, pregnancy and live birth rates with frozen epididymal and testicular sperm from obstructed (OA) and non-obstructed (NOA) patients.</p> <p>Methods</p> <p>Paternal effect of sperm source on zygote formation, embryonic cleavage, and genomic activation were examined. Additional outcome parameters monitored were clinical pregnancy rate (CPR), implantation rate (IR) and live birth rate.</p> <p>Results</p> <p>In this report, we retrospectively analyzed 156 ICSI cycles using cryopreserved epididymal sperm (ES; n = 77) or testicular sperm (TESE; n = 79). The developmental potential of embryos did not appear to be influenced by the type of surgically retrieved sperm. The average number of blastomeres observed on Day 3 was not different among different groups; 7.5 +/- 1.7 (ES), 7.6 +/- 2.1 (TESE-OA) and 6.5 +/- 2.3 (TESE-NOA). Compaction and blastulation rates, both indicators of paternal genomic activation, were similar in embryos derived from ICSI with ES or TESE from OA as well as NOA men. The only parameter significantly affected in NOA-TESE cases was the fertilization rate. CPR and IR with cryopreserved TESE (TESE-OA 59%, 34%, and TESE-NOA 37%, 20%) were also not statistically different, from that achieved with cryopreserved ES (61% and 39%). Live birth rates also appeared to be independent of sperm type. The 87 clinical pregnancies established using cryopreserved TESE and ES, resulted in the birth of 115 healthy infants. No congenital anomalies were noted.</p> <p>Conclusion</p> <p>Zygotic activation seems to be independent of sperm origin and type of azoospermia.</p

Clinical Effectiveness of Modified Laparoscopic Fimbrioplasty for the Treatment of Minimal Endometriosis and Unexplained Infertility

Objective. To study the reproductive outcomes of modified laparoscopic fimbrioplasty (MLF), a surgical technique designed to increase the working surface area of the fimbriated end of the fallopian tube. We postulated that an improvement in fimbrial function through MLF will improve reproductive outcomes. Design. Retrospective cohort study. Setting. Academic tertiary-care medical center. Patients. Women with minimal endometriosis or unexplained infertility, who underwent MLF during diagnostic laparoscopy (n=50) or diagnostic laparoscopy alone (n=87). Intervention. MLF involved gentle, circumferential dilatation of the fimbria and lysis of fimbrial adhesions bridging the fimbrial folds. Main Outcome Measures. The primary outcome was pregnancy rate and the secondary outcome was time to pregnancy. Results. The pregnancy rate for the MLF group was 40.0%, compared to 28.7% for the control group. The average time to pregnancy for the MLF group was 13 weeks, compared to 18 weeks for the control group. The pregnancy rate in the MLF group was significantly higher for patients ≤35 ys (51.5% versus 28.8%), but not for those >35 ys (17.6% versus 28.6%). Conclusion. MLF was associated with a significant increase in pregnancy rate for patients ≤35 ys