Novel method for the cryopreservation of testicular sperm and ejaculated spermatozoa from patients with severe oligospermia: a pilot study

OBJECTIVE: To investigate Volvox globator as an easy-to-handle vehicle and as a safe alternative for cryopreservation of functional motile sperm cells. DESIGN: Prospective, controlled, clinical pilot study. SETTING: Two in vitro fertilization (IVF) outpatient clinics for reproductive medicine. PATIENT(S): Fifteen patients with severe male infertility (density <100 motile sperm per milliliter) who were recruited from two IVF programs. The sperm cells were not intended for clinical use after thawing. INTERVENTION(S): In each case, a predetermined number (n = 8) of motile and morphologically intact sperm cells were injected into each Volvox sphere and then cryopreserved. The quality of the sperm cells and the handling of the Volvox spheres were verified. MAIN OUTCOME MEASURE(S): Postthaw recovery rate in cases of severe male infertility and the amount of motile sperm after thawing. RESULT(S): The postthaw recovery rate was 100%. At least 60% of the sperm cells were motile after thawing. CONCLUSION(S): The use of the spherical algae Volvox globator offers a promising, inexpensive, and easy approach to the cryopreservation of functional motile sperm cells. Volvox globator is an alternative in countries that prohibit the destructive use of oocytes, even after fertilization has failed.     

Cryopreservation of human spermatozoa: comparison of two cryopreservation methods and three cryoprotectants

OBJECTIVE: To evaluate the ability of two cryopreservation methods and three cryoprotectants to preserve sperm quality. DESIGN: A prospective clinical study. SETTING: Male infertility clinic at a tertiary healthcare center. PATIENT(S): Twenty infertile men and 10 healthy donors. INTERVENTION(S): In the first experiment, semen was cryopreserved by either the Irvine Scientific method (IS) or the Cleveland Clinic Foundation (CCF) method. In the second experiment, semen was cryopreserved by the IS method and one of three cryoprotectants: TES and Tris yolk buffer, Sperm Freezing Medium, or Enhance Sperm Freeze. MAIN OUTCOME MEASURE(S): Postthaw sperm motility, cryosurvival, and kinematics. RESULT(S): Percentages of postthaw sperm motility and cryosurvival were higher in the IS cryopreservation method compared with in the CCF method (15.94 +/- 9.19 vs. 12.07 +/- 7.31 and 47.42 +/- 17.44 vs. 35.76 +/- 17.56). However, the CCF method resulted in significantly better sperm kinematics. Postthaw motility in the donors and patients was highest in the samples frozen in TES and Tris yolk buffer medium. CONCLUSION(S): The IS method was associated with more flash freezing compared with the CCF method and resulted in better preservation of sperm motility and a higher cryosurvival rate. TES and Tris yolk buffer was most effective at protecting sperm from the negative effects of the cryopreservation process. This may be due to the presence of egg yolk along with glycerol.     

Evaluation of chromatin integrity in human sperm using acridine orange staining with different fixatives and after cryopreservation

Staining of cells with acridine orange (AO) has been widely accepted as a predictor of DNA damage in many cell types. Because of variability of protocols used in previous studies, the AO staining technique has not been widely accepted as a screening test to predict DNA damage in human sperm. In order to further validate the use of AO staining, sperm were evaluated using numerous variations in the staining protocol. This study also elucidated the effects of cryopreservation on sperm DNA. Sperm fixation in Carnoy's solution showed significantly (P < 0.05) more DNA damage (29.9 +/- 4.5%) than 2% glutaraldehyde (14.4 +/- 2.1%), 4% paraformaldehyde (5.5 +/- 1.7%), no fixation (15.8 +/- 4.3%) but did not differ from Diff Quik solution (19.2 +/- 5.8%). No difference was observed for sperm DNA damage assessment using a 0.2 m (15.5 +/- 3.2%) or 0.3 m (14.9 +/- 3.3%) concentration of Na(2)HPO(4).7H(2)O in the AO staining solution. Frozen-thawed semen samples showed increased damage to sperm DNA under both Carnoy's (fresh: 10.9 +/- 1.3%; frozen: 30.8 +/- 2.9%; P < 0.05) and Diff Quik fixation (fresh: 6.2 +/- 0.8; frozen: 17.1 +/- 2.5%P < 0.05). Present data also showed that spermatozoa from some individuals are more prone to DNA damage after freezing and thawing procedures than others. In conclusion, Carnoy's fixative provides a better predictive value for DNA damage to sperm using AO staining. Additionally, cryopreservation increased damage to the sperm DNA.     

When to avoid creating surplus human embryos

The advice that should be given to a couple considering assisted reproductive technologies for the treatment of their infertility, when they are completely opposed to the destruction of surplus embryos, is discussed. It is urged that they do not use treatments that generate surplus embryos. They should be given the options of declining IVF and considering adoption, or less efficient treatments, namely limited ovarian stimulation, limited insemination of available ova or natural cycle IVF where no surplus embryos are generated.     

Recovery of fertility after grafting of cryopreserved germinative tissue in female rabbits following radiotherapy

BACKGROUND: Many cancer survivors face infertility as a consequenceof the aggressive treatment they must undergo. Cryopreservationof ovarian tissue before chemotherapy or radiotherapy may allowfor tissue transplantation after the treatment, and restorationof fertility. We tested the potential of an orthotopic autograftingof cryopreserved germinative tissue in female rabbits with ovarianfailure following radiotherapy. METHODS: Ten adult multiparousfemale rabbits were randomly allocated into two groups, fivein group I (control) and five in group II (transplant). Allrabbits underwent right oophorectomy with cryopreservation ofthe germinative tissue, followed by sterilization of the remainingleft ovary by radiotherapy. Later, group II rabbits receivedin the irradiated left ovary an implant of the frozen germinativetissue from the right ovary, whose small pieces were freelyspread intracortically in a procedure we named ‘intracorticalsowing of germinative tissue’ (ISGT). RESULTS: All groupII rabbits conceived following spontaneous mating within 6 monthsof the transplant, whereas none of the remaining rabbits ingroup I had conceived up to 11 months after transplant. CONCLUSIONS:This study suggests that fertility can be restored in rabbitsby sowing cortical tissue in a previously irradiated ovary.The clinical feasibility of this technique remains to be determined.     

Cryopreservation of metaphase II human oocytes effects mitochondrial membrane potential: implications for developmental competence

BACKGROUND: Current outcome results with embryos derived from thawed MII human oocyes are significantly lower than with embryos cryopreserved at the pronuclear stage. Here, we investigated whether freezing-thawing was associated with changes in oocyte mitochondrial polarity (DeltaPsim) that could influence competence by altering ATP levels or the ability of the cytoplasm to regulate intracellular Ca2+. METHODS: Fresh and thawed uninseminated and unfertilized MII oocytes were stained with the DeltaPsim-specific probe JC-1 to detect clusters of high-polarized mitochondria (J-aggregate positive) and with the Ca2+- specific probe Fluo-4 to measure changes in intracellular levels of this cation. ATP content per oocyte was measured directly and cortical granules were visualized with a cortical granule-specific probe. RESULTS: A significant difference between fresh and thawed MII oocytes existed for pericortical J-aggregate fluorescence and for the ability of the cytoplasm to increase free Ca2+ in response to ionophore exposure. No significant difference in ATP contents was measured and cryopreservation was not associated with an apparent release of cortical granules. CONCLUSION: Irreversible loss of high DeltaPsim in thawed oocytes may be associated with defects in Ca2+ signalling after insemination and could have downstream consequences for normal embryogenesis.     

Mouse Ovarian Tissue Cryopreservation Has Only a Minor Effect on In Vitro Follicular Maturation and Gene Expression

Purpose : To establish a protocol for ovarian tissue cryopreservation which can retain fertility potential after thawing and to evaluate the impact of cryopreservation on development and gene expression during folliculogenesis. Methods : A controlled randomized study in a clinical and academic research setting in a university medical center was conducted to study cryopreservation and in vitro maturation (IVM) of mouse ovarian follicles. Preantral follicles isolated from either fresh (Group A) or cryopreserved (Group B) murine ovarian tissues were used to test their fertility potential by in vitro culture–in vitro maturation (IVC-IVM). Expression of Graafian follicles derived from both groups were detected by DNA microarray techniques for comparison. Results : Although there were no significant differences in IVM outcomes and follicular gene expression between the two experimental groups, cryopreservation appears to induce the expression of heat shock proteins, DNA-damage-inducible protein 45 and death-related apoptosis genes (i.e., Fas and Fas-ligand). Conclusion : Cryopreservation may trigger biological events not amenable to normal cell function and follicular development. However, neither follicular development nor gene expression was dramatically changed after cryopreservation. These data suggest that although our current cryopreservation techniques yield competent follicles and mature oocytes, subtle changes observed in gene expression imply that the present cryopreservation techniques need to be further refined.     

Oocyte maturation,follicle rupture and luteinization in human cryopreserved ovarian tissue following xenografting

BACKGROUND: Previous studies have demonstrated development of antral follicles in cryopreserved human ovarian tissue after autografting and xenografting, thus indicating successful preservation of follicular function. The study aim was to assess whether these follicles could also undergo periovulatory changes in response to hCG. METHODS: Ovarian tissue from three patients were dehydrated in propanediol (PROH)/sucrose and cryopreserved using the slow cooling/rapid thaw procedure. Thawed tissue was placed under the kidney capsule in immunodeficient mice. Following growth (>20 weeks) in the presence of gonadotrophin, hCG was administered and ovarian tissue examined histologically. RESULTS: Thirty-two antral follicles (diameter range 0.6 to 5 mm) were examined. Histological evidence of a response to hCG was evident in all follicles. Disruption of the concentric layers of mural granulosa and theca cells was apparent in all antral cavities. In 17 (53%) follicles the exterior follicular wall had reduced to a few cells thick, and in eight (25%) the wall had ruptured. Mucified oocyte-cumulus cell complexes were present in 32 follicles, 17 of which had begun to detach from the pedicle. Resumption of meiosis had occurred in over half the oocytes (five metaphase II and seven metaphase I oocytes, eight germinal vesicle breakdown). Two corpora lutea were also detected. CONCLUSIONS: Follicles cryopreserved within human ovarian tissue using the PROH procedure, can develop to the antral stage and undergo periovulatory changes following xenografting and exposure to a luteinizing stimulus.     

Birth of a healthy baby after transfer of embryos that were cryopreserved for 8.9 years

OBJECTIVE: To report successful pregnancy from embryos that had been cryopreserved for 8.9 years. DESIGN: Case report. SETTING: Fertility clinic of the University of Buenos Aires School of Medicine. PATIENT(S): A 43-year-old woman with ovarian failure and a 45-year-old man with nonobstructive azoospermia underwent embryo donation.I NTERVENTION(S): Five embryos that had been cryopreserved and stored since 1989 were thawed in 1998 and donated to an infertile couple. Endometrial preparation was performed with 17beta-estradiol and progesterone. The four embryos with better morphologic characteristics were transferred into the uterus. MAIN OUTCOME MEASURE(S): Post-thaw embryo survival, pregnancy, and birth. RESULT(S): Embryo survival was satisfactory as assessed by morphology. Pregnancy was confirmed by ultrasonography. A healthy baby weighing 2120 g was delivered by cesarean section at 36 weeks. CONCLUSION(S): Human 8-cell embryos may be viable after extended storage and can result in successful pregnancy.     

Donor oocyte cytoplasmic transfer did not enhance implantation of embryos of women with poor ovarian reserve

Purpose : To determine whether donor oocyte cytoplasm transferred into the oocytes of women 40 years or with diminished ovarian reserve would enhance embryo quality, implantation, or pregnancy rates. Methods : Study subjects included women 40 years (15) or with abnormal FSH levels (3). Healthy volunteers (18) produced oocytes for cryopreservation. Donor oocytes were thawed and cytoplasm from surviving oocytes was injected with a single sperm into the cytoplasm of recipient oocytes. Outcome measures included embryo quality scores, implantation, and pregnancy rates. Results : Eighteen donors produced 213 oocytes for cryopreservation and 39/171 (22.8%) survived thawing. Eighteen recipients initiated 25 IVF cycles with embryo transfer in 20 cycles after cytoplasmic transfer (CT). Four cycles resulted in three biochemical losses and one aneuploid clinical loss. Embryo quality did not improve with CT compared to pre-CT IVF cycles in six recipients. Conclusions : CT with cryopreserved donor oocyte cytoplasm did not enhance success in women with advanced reproductive age or low ovarian reserve.