Effect of different cryopreservation protocols on the metaphase II spindle in human oocytes

The aim of this study was to evaluate the impact of different cryopreservation protocols on the repolymerization of metaphase (M)II spindles in human oocytes. Fresh aspirated donor oocytes were cryopreserved 3-4 h after retrieval using four different protocols: slow freezing using 1.5 mol/l 1,2-propanediol (PROH) + 0.2 mol/l sucrose (n = 36); 1.5 mol/l PROH + 0.3 mol/l sucrose (n = 34); 1.5 mol/l PROH + 0.3 mol/l sucrose with Na(+) depleted-choline replaced media (n = 27), and vitrification by the Cryotip method (n = 23). The control group comprised 34 fresh oocytes. Three hours after thawing, surviving and control oocytes were fixed for meiotic spindle/chromatin assessment. Survival rates were 63.8, 73.5, 74.1 and 86.9% respectively for the four protocols described above. Survival for vitrified oocytes was higher than that observed for slow freezing with 0.2 and 0.3 mol/l sucrose (P < 0.05). The proportion of oocytes showing normal spindle configuration was similar for the four protocols (81, 73.9, 88.9 and 81.3% respectively) and 88.5% for controls, showing that the MII spindle returns to its normal configuration after 3 h of post-thawing incubation under standard conditions, irrespective of the cryopreservation technique used.     

Cytoskeleton and polyploidy after maturation and fertilization of cryopreserved germinal vesicle—stage mouse oocytes

Purpose Our purpose was to assess the effect of cryopreservation on cytoskeleton of germinal vesicle (GV) mouse oocytes and determine whether irreversible spindle damage and related digyny associated with cryopreservation of metaphase II (MII) oocytes can be avoided. Methods The GV oocytes were cryopreserved using a slowcooling (0.5‡C/min) and slowthawing (8‡C/min) protocol in 1.5 M dimethylsulfoxide supplemented with 0.2 M sucrose and analyzed before and during fertilization by multiplelabel fluorescence and differential interference contrast microscopy techniques. Results When examined after in vitro maturation, the vast majority (>95%) of cryopreserved and control oocytes displayed normal microfilament and microtubule organization. With respect to barrelshaped spindle and normal chromosome alignment, no significant differences were observed between cryopreservation (78 and 86%, respectively) and control (85 and 95%, respectively) groups. In fertilization experiments, spindle rotation, formation of the second polar body, and pronuclear migration were displayed by similar percentages of cryopreserved (96, 94, and 37%, respectively) and control (98, 97, and 45%, respectively) oocytes, indicating normal functionality of the cytoskeleton during this period. However, pronuclear formation was significantly inhibited by cryopreservation (81%) compared with controls (100%). Regarding digyny and polyspermy, no significant increase was observed after cryopreservation (3 and 10%, respectively) compared with controls (3 and 6%, respectively). Conclusions Cryopreservation of mouse oocytes at the GV stage is particularly advantageous to circumvent the spindle damage and increased digyny noted after cryopreservation of MII oocytes.     

A PolScope evaluation of meiotic spindle dynamics in frozen–thawed oocytes

In mature human oocytes, the metaphase II (MII) spindle presence and birefringence signal detected through the PolScope may vary before and after freezing. In particular, spindle dynamics during the first few hours after thawing is still under study. In this study, oocytes from stimulated ovaries were cryopreserved in 1.5 mol/l 1,2-propanediol with 0.3 mol/l sucrose using a slow freezing–rapid thawing method. Oocytes were examined with the PolScope for the presence, intensity of signal birefringence and size of the meiotic spindle before freezing and at 0, 1 and 2 h post-thaw (where 0 h = the time of the end of the thawing procedure). Of the 173 surviving oocytes exhibiting a spindle before freezing, 82.7% (143/173) showed spindle birefringence within 1 h of thawing. However, at the end of the thawing procedure the intensity of spindle birefringence (retardance) and the spindle length were smaller in comparison to the pre-freezing condition. These parameters increased after 1 h, although were not restored to the values observed before freezing. No significant changes were observed by extending the culture to 2 h.     

Blastocyst Development After Cryopreservation and Subcutaneous Transplantation of Mouse Ovarian Tissue

Purpose To investigate follicle survival and developmental potential with IVF of cryopreserved, subcutaneously transplanted mouse ovarian tissue.Methods Fresh and frozen mouse ovarian tissue was autologously transplanted into subcutaneous tissue. Two weeks after the transplantation, the morphology and histology of the fresh and frozen grafts were compared. Superovulation and IVF was performed to evaluate the fertility potential of the frozen ovarian graft.Results Both fresh and frozen grafts of ovarian tissue survived in 14 of 16 mice (88%). Morphologically, both types of grafts resembled fresh ovarian tissue and contained follicles at all stages of folliculogenesis. A total of 73% of follicles in fresh grafts and 62% in frozen grafts survived after transplantation compared with fresh ovarian tissue. Sixteen ICR mice underwent superovulation. A total of 56 oocytes from antral follicles were recovered from the subcutaneously transplanted cryopreserved ovarian tissue. Fourteen (25%) oocytes were in metaphase II stage, 6 were fertilized by IVF, and 2 progressed to the blastocyst stage.Conclusions Cryopreservation and subcutaneous transplantation of ovarian tissue provides a possible means of fertility preservation. The main loss of follicles occurred during grafting rather than during freezing and thawing.     

Cryopreserved Immature Mouse Oocytes: A Chromosomal and Spindle Study

Purpose: Cryopreservation of human oocytes might provide an alternative approach to freezing supernumerary embryos obtained during IVF. This process, performed on immature denuded prophase I mouse oocytes, was investigated. Methods: We first investigated the capacity of frozen, immature, murine oocytes to continue in vitro maturation after thawing. We then evaluated the risk to offspring from chromosomal damage by cytogenetical and cytological (spindle) analysis. Finally, we attempted to determine the reasons for and the stage of maturation failure. Results: A total of 700 immature oocytes was frozen, 629 (90%) were recovered intact after thawing, and 53% extruded the first polar body, versus 74% for the control group. Freezing was not accompanied by an increase in aneuploidy in maturing oocytes (18 and 15% for thawed and control oocytes, respectively). Consequently, the first meiotic division occurred normally, without an increase in nondisjunction. Spindle analysis demonstrated only a few abnormalities (15 and 2% for thawed and control oocytes, respectively) incompatible with further development. Oocytes arrested during in vitro maturation were mainly at the metaphase I stage (64 and 76% for thawed and control oocytes, respectively). Whereas 17% of thawed oocytes were blocked before the formation of the first meiotic spindle, this never occurred in the control group. Conclusions: Immature murine oocytes can withstand cryopreservation, which is encouraging for future human application of this technique.     

Letters to the editor

Objective: To describe the birth achieved from frozen embryos after intracytoplasmic sperm injection (ICSI) of donor sperm into vitrified oocytes.

Patient: A 25-year-old woman whose husband was azoospermic undergoing IVF therapy.

Methods: Oocytes collected after ovarian stimulation were vitrified, thawed, and fertilized by frozen donor sperm.

Results: Nineteen oocytes were vitrified and all survived after thawing. Thirteen of the 19 oocytes that underwent ICSI with donors sperm were successfully fertilized. Twelve embryos were cryopreserved again by conventional slow-freezing protocol because of uterine bleeding on the day of transfer. Three thawed embryos were transferred, and a normal male with an infant karyotype of 46,XY was delivered.

Conclusion: This case report demonstrates effective oocyte cryopreservation by vitrification.     

Mouse oocytes injected with cryopreserved round spermatids can develop into normal offspring

Purpose: This study was performed to determine whether frozen-thawed mouse round spermatids can fertilize oocytes and contribute to normal embryo development. Methods: Freshly collected mouse testicular cells were frozen in PBS containing 7.5% glycerol and 7.5% fetal bovine serum. After thawing and removal of the cryoprotectants, round spermatids were selected and injected individually into mature oocytes which had been previously activate with Sr ²⁺ -containing Ca ²⁺ -free medium. Results: After thawing, 75–85% of testicular cells were alive. About 90% of the oocytes were fertilized by intracytoplasmic injection of frozen-thawed round spermatids; 11% (17/150) of embryos transferred to foster mothers developed into normal offspring. Conclusions: Mouse round spermatids can be cryopreserved for production of normal offspring.     

Cryopreservation of immature and in-vitro matured human oocytes by vitrification

The objective of this study was to determine the efficacy of vitrification of human oocytes before and after in-vitro maturation (IVM). The immature oocytes recovered (n = 472) were divided into two groups: (i) immature oocytes (n = 219) vitrified at the germinal vesicle (GV) stage; and (ii) immature GV-stage oocytes (n = 253) that were firstly matured in vitro (MII-stage oocytes; n = 178), then vitrified (n = 79). The remaining oocytes (n = 99), which were not vitrified, were processed as controls. After warming, the oocyte survival, maturation and fertilization rates, as well as embryonic development, were compared. The results showed no significant difference between the survival rates of the oocytes vitrified at GV stage and those vitrified at MII stage (85.4% versus 86.1%). However, oocyte maturation rates were significantly reduced (P < 0.05) when oocytes were vitrified at immature GV stage followed by IVM (50.8%) in comparison with the control group (70.4%). Following insemination by intracytoplasmic sperm injection, there was no difference in the fertilization (62.1% versus 58.8%), cleavage (69.5% versus 67.5%) and blastocyst development (0.0% versus 0.0%) rates between these two groups. However, these results were significantly lower (P < 0.05) than those achieved in the control group. This suggests that better results can be achieved by vitrifying mature oocytes rather than immature oocytes.     

Successful delivery following cryopreservation of zygotes produced by in vitro matured oocytes retrieved from a woman with polycystic ovarian syndrome-like disease: a case report

Purpose : To estimate frozen zygotes, which developed from in vitro matured oocytes retrieved from polycystic ovarian syndrome-like disease. Methods : Oocyte retrieval was performed on Day 15 following withdrawal bleeding. The oocytes were incubated for 24 h in TCM-199 maturation medium supplemented with follicle fluid, E2, FSH, and hCG. Results : A total of 12 immature oocytes were collected. Seven of the 12 oocytes (58.3%) developed to the metaphase-II stage, and subsequently, all seven fertilized oocytes were frozen at the pronuclear stage. The remaining five oocytes failed to develop to the metaphase-II stage after an additional 24 h of incubation. Three of seven cryopreserved oocytes were thawed and developed to 2–8-cell cleaved stage embryos. The first pregnancy failed. However, the second frozen–thawed embryo transfer resulted in the delivery of healthy twins. Conclusions : Successful delivery using frozen zygotes from an anovulatory woman with polycystic ovarian syndrome-like disease.     

Successful Pregnancy After the Vitrification of Zygotes Using Commercial Vitrification Solutions and Conventional Straws to Protect Against Infections in Liquid Nitrogen

Purpose: To report on successful birth after the transfer of postthawed human zygotes that were vitrified using a conventional straw for the purpose of protecting them from infections and a low-toxicity cryoprotectant that is commercially sold.Methods: A primary infertile couple presented at our IVF program. After being checked for fertilization, the embryos were not transferred to the uterus at that cycle. Instead, all of them were cryopreserved at the 2-pronuclei stage using our original vitrification method. After the vitrification and warming of four zygotes, two embryos were transferred into the uterus.Results: Twenty-one 2-pronuclei embryos were vitrified in liquid nitrogen. After 2 embryos were thawed and transferred, successful pregnancy was the outcome, and a healthy boy was born at term.Conclusions: Vitrification is a simple procedure and requires less time than slow freezing. Vitrification of zygotes in a conventional straw seems to be sufficient for viability and works to store the zygotes safely.     

Cryopreservation of hamster oocytes: effects of vitrification or freezing on human sperm penetration of zona-free hamster oocytes

Three experiments were conducted for evaluation of the efficacy of conventional freezing or vitrification of hamster oocytes for use in a human sperm penetration assay (hSPA). In experiment 1, oocytes were cryopreserved and evaluated for survival on the basis of morphologic criteria. Survival of vitrified oocytes and that of frozen oocytes were not different, whereas all cryopreserved groups had lower survival than noncryopreserved controls. In experiment 2, oocytes were conventionally frozen or vitrified and evaluated in an hSPA. Vitrified oocytes had a lower frequency of sperm penetration than frozen oocytes, and all cryopreserved groups had lower penetration rates than untreated controls. In experiment 3, oocytes were exposed to the cryoprotectant used to vitrify (VS1) or freeze (DMSO) but not cooled prior to evaluation in an hSPA. Exposure to DMSO but not VS1 reduced hSPA values. It is concluded from these experiments that while all cryopreserved oocytes do not survive, at current stages of development conventionally frozen oocytes perform better than vitrified oocytes in the hSPA and losses associated with conventional freezing procedures may be related to cryoprotectant exposure, whereas vitrification losses are more probably due to events associated with rapid cooling and/or warming of the oocytes.     

Selective vitrification of euploid oocytes markedly improves survival, fertilization and pregnancy-generating potential

Enthusiasm for oocyte cryopreservation has been limited by poor pregnancy rates per thawed metaphase II (MII) oocytes (<4%) and low implantation rates per embryos. The reasons relate to technical limitations in the freezing process, and the fact that <40% of oocytes are euploid and unable to produce 'competent' embryos. Comparative genomic hybridization was performed on the first polar body (PB-1) of 323 MII oocytes retrieved from 16 donors. Of these, 111 were euploid, and were vitrified. Seventy-five of 78 vitrified oocytes (96%) survived warming and were fertilized using intracytoplasmic sperm injection. Thirty-one (41%) subsequently developed into expanded blastocysts, of which no more than two were subsequently transferred per uterus to 16 out of 19 prospective embryo recipients. Twelve of 19 (63%) recipients produced 17 healthy babies (eight singletons, three twins, and one set of triplets) One twin pregnancy miscarried in the late first trimester The birth rate per transfer of a maximum of two blastocysts to 16 recipients was 75%. The implantation rate per vitrified euploid oocyte was 27%. This study showed a six-fold improvement in pregnancy rate per cryopreserved oocyte over previous reports and a marked improvement in implantation rate. If independently validated, this approach could open the door to commercial egg cryobanking, significantly expanding women's reproductive choices.     

Fertility preservation for trans men: frozen-thawed in vitro matured oocytes collected at the time of ovarian tissue processing exhibit normal meiotic spindles

Purposes

At the moment of sex reassignment surgery (SRS), the ovarian tissue is sometimes cryopreserved as fertility preservation option for female-to-male trans men, also called trans men. During this preparation, cumulus-oocyte-complexes (COCs) can be found and in vitro matured. It is not known if these oocytes are developmentally competent. In order to use these oocytes for fertility preservation and subsequent fertilization, a normal spindle structure before and after vitrification is necessary.

Methods

A total of 680 COCs were collected from trans men (n = 16) at the time of SRS and after testosterone treatment. The COCs were subjected to in vitro maturation and those that reached the metaphase II stage (MII) were collected and split into two groups; group 1 was immediately fixed for spindle staining and group 2 was first vitrified and warmed followed by spindle staining. Statistical analysis was performed by Fisher’s exact test.

Results

After 48 h in vitro maturation, 38.1% of COCs were at MII stage. Those oocytes were split in two groups: (1) 126 MII oocytes in the noncryopreservation group and (2) 133 MII oocytes underwent cryopreservation through vitrification. The oocyte survival rate, after 2 h warming, was 67.7%. Both the noncryopreserved and the vitrified group showed comparable results concerning normal spindle structure and chromosomes alignment, 85.7% vs. 92.2% (P = 0.27).

Conclusions

Spindle structure analysis and chromosomal alignment after vitrification seem normal in in vitro matured COCs collected during the tissue processing of ovaries in trans men at the time of SRS. The MII oocytes do not seem to be morphologically affected by prolonged testosterone treatment.

     

Freeze/thaw stress induces organelle remodeling and membrane recycling in cryopreserved human mature oocytes

Purpose

Our aim was to evaluate the ultrastructure of human metaphase II oocytes subjected to slow freezing and fixed after thawing at different intervals during post-thaw rehydration.

Methods

Samples were studied by light and transmission electron microscopy.

Results

We found that vacuolization was present in all cryopreserved oocytes, reaching a maximum in the intermediate stage of rehydration. Mitochondria-smooth endoplasmic reticulum (M-SER) aggregates decreased following thawing, particularly in the first and intermediate stages of rehydration, whereas mitochondria-vesicle (MV) complexes augmented in the same stages. At the end of rehydration, vacuoles and MV complexes both diminished and M-SER aggregates increased again. Cortical granules (CGs) were scarce in all cryopreserved oocytes, gradually diminishing as rehydration progressed.

Conclusions

This study also shows that such a membrane remodeling is mainly represented by a dynamic process of transition between M-SER aggregates and MV complexes, both able of transforming into each other. Vacuoles and CG membranes may take part in the membrane recycling mechanism.

     

Successful live birth from oocytes after more than 14?years of cryopreservation

ObjectiveTo report a birth of a healthy girl after long-term oocyte cryopreservation by slow cooling in sodium depleted medium.DesignClinical application.SettingUniversity Affiliated, Private IVF center.PatientA 38-year-old woman received embryos from IVF by intracytoplasmic sperm injection (ICSI) with her own oocytes that were cryopreserved by slow freezing in a low-sodium medium 14 years and 6 months before, when she was 24 years old.Result(s)From six metaphase-II oocytes thawed, two survived, one was fertilized after ICSI and a cleaving embryo was transferred on day 3. A single term pregnancy was achieved, ending with the delivery of a healthy girl.Conclusion(s)Cryopreservation after slow freezing in a sodium depleted medium maintained the developmental competence of oocytes after long-term storage and resulted in a successful live birth. As far as is known, this case represents, up to date, the longest storage period of cryopreserved human oocytes resulting in a live birth.     

Deliveries of babies with normal health derived from oocytes with smooth endoplasmic reticulum clusters

PurposeTo examine the impact on development of derived embryos from smooth endoplasmic reticulum clusters (SERC) in human metaphase II (MII) oocytes.MethodsRetrospective analysis at Kyono ART Clinic. Comparison of embryological development, pregnancy, live birth and fetal malformation between oocytes with SERC (the SERC(+) group) and those without (the SERC(−) group) in 2,158 patients (3,758 cycles) after ICSI.ResultsFertilization and implantation rate were significantly lower in SERC(+) MII oocytes than in SERC(−) MII oocytes. After the transfer of fresh and vitrified embryos derived from SERC(+) oocytes, 14 pregnancies resulted in 14 healthy babies, including 2 from fresh embryo transfer (ET) and 12 from vitrified-warmed ET, with no malformations.Conclusion(s)The presence of SERC in MII oocytes was associated with significantly lower fertilization rates and implantation rates than seen in SERC(−) MII oocytes within SERC (+) cycles. However, SERC had no impact on post-implantation development as well as neonatal outcome.     

Comparison of Pregnancy Outcome of Pronuclear- and Multicellular-Stage Frozen–Thawed Embryo Transfers

Purpose: Our purpose was to determine if supernumerary embryos generated by in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) should be frozen (using 1,2-propanediol) at the pronuclear or multicellular stage. Methods: The study was a retrospective analysis conducted at the Dubai Gynaecology & Fertility Centre of the Department of Health & Medical Services, Dubai, U.A.E. One hundred forty-one women undergoing frozen–thawed embryo replacement cycles with IVF generated embryos and 84 women undergoing the same with ICSI generated embryos. Results: Supernumerary, IVF-generated embryos frozen at the multicellular stage had a significantly higher rate of survival on thawing (73.9%) than embryos frozen at the pronuclear stage (64.4%). The morphological grades of the embryos in the two groups were similar, but a significantly higher pregnancy rate was obtained with embryos frozen at the multicellular stage (22.8%) than with pronuclear-stage embryos (14.8%). Similarly, with ICSI-generated embryos, significantly higher survival was seen with multicellular-stage frozen embryos (74.8%) than pronuclear-stage embryos (64.4%). The morphological grades of the embryos and pregnancy outcomes of the two groups were similar. Conclusions: Supernumerary embryos generated by IVF and ICSI should be frozen at the multicellular stage so as to allow selection of the best embryos for transfer and embryo freezing of only robust embryos.     

The freezing method of cleavage stage embryos has no impact on the weight of the newborns

Purpose

The aim of this study was to study the effect of the embryo freezing method on the birth weight of newborns from frozen embryo transfer (FET) cycles, and the pregnancy results of cleavage stage embryos cryopreserved by slow freezing or vitrification.

Methods

This is a retrospective cohort study undertaken in a University Hospital IVF unit using concurrently both the slow-freezing and the vitrification techniques. All frozen-thawed and vitrified-warmed day 2 and day 3 embryo transfers during the time period from 1 April 2009 to 31 November 2013 were included in the study.

Results

There was no statistically significant weight difference between newborns from vitrified or slow-frozen embryos (3588 vs 3670 g). A higher post-thaw viability rate was achieved after cryopreservation by the vitrification technique compared to the slow-freezing protocol (83.4 vs 61.4 %). The miscarriage rate was lower in the vitrification group (15.7 vs 29.0 %). The live birth rates were similar (19.5 vs 19.1 %) in the slow-freezing and vitrification groups, respectively. Among vitrified embryos, 7.4 embryos needed to be thawed to produce one delivery; in the slow-freezing group, that number was 11.9.

Conclusions

The freezing method has no impact on the weight of the newborn.With lower post-thaw survival rates and higher miscarriage rates, the slow-freezing cryopreservation protocol is inferior to the vitrification technique.

     

Successful freezing of unfertilized mouse oocytes and effect of cocultures in oviducts on development of in vitro fertilized embryos after thawing

Purpose To establish a freeze-thawing method for unfertilized oocytes with a high success rate, we examined several conditions for freeze-thawing. The effects of EDTA and cocultures in oviducts on the development of embryos fertilized in vitro after thawing were also studied. Results In the first experiment, unfertilized oocytes that were frozen in 1.5 Mdimethylsulfoxide (DMSO) supplemented with 0.2 Msucrose by a slow freeze-thawing method showed the best results (fertilization rate, 71.9%; blastocyst rate per frozen oocyte, 18.8%). The proportion of embryos that developed to blastocysts was significantly higher when DMSO was added at 4‡C than at room temperature (39.4 vs 19.4%; P<0.01). The addition of EDTA (10 ΜM)to the culture medium did not promote embryo development after fertilization in vitro. However, the rate of development of in vitro fertilized embryos to blastocysts after thawing was significantly higher when the embryos were cultured in oviducts in vitro than the rates in control cultures and those cultured with EDTA (blastocyst rate from fertilized oocytes, 71.4 vs 51.0 and 52.8%, respectively; P<0.01). Conclusion Unfertilized mouse oocytes can be cryopreserved successfully by a slow freeze-thawing method with the addition of 1.5 MDMSO and 0.2 Msucrose at low temperatures, and coculture with oviducts enhances the development of embryos that are fertilized in vitro after thawing.     

Pronuclear stage cryopreservation after intracytoplasmic sperm injection and conventional IVF: implications for timing of the freeze

Objective: To compare clinical outcomes of frozen embryo transfers using cryopreserved pronuclear stage oocytes that had undergone either intracytoplasmic sperm injection (ICSI) or conventional IVF.

Design: Observational.

Setting: A tertiary referral reproductive medicine unit.

Patient(s): Couples undergoing either ICSI or conventional IVF from January 1, 1995 to December 31, 1997.

Intervention(s): Patients underwent a standard controlled ovarian hyperstimulation protocol and transvaginal ultrasound-guided oocyte retrieval. All normally fertilized (2PN) oocytes exceeding a specified embryo number designated for fresh transfer were immediately cryopreserved at the pronuclear stage. Our cryopreservation method included timing of the freeze according to pronuclear morphology. Subsequent frozen embryo thaw-transfer cycles were usually performed by thawing only the intended number of embryos for transfer.

Main Outcome Measure(s): Thaw survival rate, implantation rate, clinical pregnancy rate, delivery rate.

Result(s): Ninety-six thaw-transfer cycles (n = 72) and 93 thaw-transfer cycles (n = 67) were undertaken in patients who had previously undergone conventional IVF or ICSI, respectively. Embryo thaw survival rates (IVF, 90.4%; ICSI, 91.1%) were similar. Clinical pregnancy (IVF, 40.6%; ICSI, 44.1%) and delivery (IVF, 36.4%; ICSI, 39.8%) rates per transfer, as well as implantation (IVF, 19.1%; ICSI, 19.9%) rates, were also similar. There were only four clinical pregnancy losses in both groups.

Conclusion(s): Embryo thaw survival is similar for cryopreserved pronuclear stage oocytes derived from ICSI and conventional IVF. Clinical pregnancy, implantation and delivery rates were also similar for the two groups. In addition, there was no increase in the rate of pregnancy loss in ICSI patients after frozen embryo transfers.