Validation of safety procedures for the cryopreservation of semen contaminated with hepatitis C virus in assisted reproductive technology

BACKGROUND: In France, assisted reproductive technologies involving a hepatitis C virus (HCV)-infected man requires the cryopreservation of potentially infected semen (in order to establish the presence of HCV), hence the need for a safe and secure storage system. We evaluated the safety of high-security straws for the conservation of semen containing HCV RNA under routine conditions. METHODS: Ionomeric resin (IR) straws were filled with seminal plasma spiked with different concentrations of HCV RNA and sealed using a thermo-solder. After a 4% sodium hypochlorite treatment and/or cryopreservation for 7 days in liquid nitrogen, the outside ends of each straw were rinsed with RNAse-free water. RESULTS: No HCV RNA could be detected in any of the water samples. Additional samples included the rinsing water from straws sealed by thermo-solder and from the heating wire used to cut the end of straws containing HCV-positive semen. The latter samples were found positive for both HCV RNA and the protamine-2 gene expressed by spermatozoa. CONCLUSIONS: These results demonstrate the safety of IR straws, the filling system and the thermo-solder for cryopreservation of semen containing HCV in liquid nitrogen. Decontamination of the straw after sealing and the use of disposable scissors to open the straws are strongly recommended.     

Birth following vitrification of a small number of human oocytes: case report

We report the birth of a healthy baby girl at 37 weeks gestation to a 47 year old recipient, after vitrification of mature oocytes from four in-vitro fertilization (IVF) patients. A total of 17 oocytes was vitrified in 1-2 microl of ethylene glycol (40%) and 0.6 mol/l sucrose (20.54%) in open pulled straws. Eleven oocytes survived after vitrification and five pronuclear zygotes were obtained after intracytoplasmic sperm injection (ICSI). Three embryos were transferred to three patients, two of whom were the original oocyte donors and pregnancy was not established. The third embryo was donated to a 47 year old infertile woman after preimplantation diagnosis had confirmed euploidy for chromosomes X, 13, 14, 15, 16, 18, 21 and 22. The successfully completed pregnancy is encouraging for further research to explore the potential benefits of vitrification for the cryopreservation of human oocytes, given the relatively low success of conventional freezing of human oocytes by slow cooling methods.     

1,2-propanediol and the type of cryopreservation procedure adversely affect mouse oocyte physiology

BACKGROUND: The aim of this work was to examine the effect of 1,2-propanediol (PrOH) and type of cryopreservation procedure (slow freezing and vitrification) on oocyte physiology. METHODS: Intracellular calcium of mouse metaphase II (MII) oocytes was quantified by fluorescence microscopy. The effect of PrOH on cell physiology was further assessed through analysis of zona pellucida hardening and cellular integrity. Protein profiles of cryopreserved oocytes were generated by time-of-flight mass spectrometry (TOF-MS). RESULTS: PrOH caused a protracted increase in calcium, which was sufficient to induce zona pellucida hardening and cellular degeneration. Using 'nominally calcium free' media during PrOH exposure significantly reduced the detrimental effects. Proteomic analysis identified numerous up- and down-regulated proteins after slow freezing when compared with control and vitrified oocytes. CONCLUSIONS: Using such approaches to assess effects on cellular physiology is fundamental to improving assisted reproduction techniques (ART). This study demonstrates that PrOH causes a significant rise in intracellular calcium. Using calcium-free media significantly reduced the increase in calcium and the associated detrimental physiological effects, suggesting that calcium-free media should be used with PrOH. In addition, analysis of the oocyte proteome following cryopreservation revealed that slow freezing has a significant effect on protein expression. In contrast, vitrification had a minimal impact, indicating that it has a fundamental advantage for the cryopreservation of oocytes.     

Effects of cryopreservation on human sperm acrosomes.

Total acrosin activity and acrosomal status were determined before and after cryopreserving human spermatozoa. Three different cryopreservation protocols were used. Both acrosin activity and the incidence of intact acrosomes decreased during cryopreservation. The magnitudes of the decreases were weakly but significantly correlated (r = 0.29, P less than 0.05), suggesting that acrosomal loss contributed to the decrease in acrosin activity. The effects of the three cryopreservation protocols were not significantly different. Motility decreased more (average 43%) than did the percentage of spermatozoa with intact acrosomes (27%) and the total acrosin activity (24%). These measurements suggested that acrosomal damage may have been secondary to cell death. This hypothesis was tested by determining the acrosomal status of spermatozoa that survived cryopreservation. Spermatozoa that were motile after thawing averaged 96% acrosome-intact; their acrosin activity, however, was significantly less than that of motile, unfrozen spermatozoa. These observations support the idea that the acrosomal loss due to cryopreservation is associated with cell death but also demonstrate decreased total acrosin activity of the acrosome-intact spermatozoa that survive cryopreservation.     

Experimental vitrification of human compacted morulae and early blastocysts using fine diameter plastic micropipettes

BACKGROUND: Vitrification of human blastocysts has been successfully applied using grids, straws and cryoloops. We assessed the survival rate of human compacted morulae and early blastocysts vitrified in pipette tips with a smaller inner diameter and solution volume than the previously described open pulled straw (OPS) method. METHODS: Excess day 5 human embryos (n = 63) were experimentally vitrified in vessels. Embryos were incubated at 37 degrees C with sperm preparation medium (SPM) for 1 min, SPM + 7.5% ethylene glycol (EG)/dimethylsulphoxide (DMSO) for 3 min, and SPM + 16.5% EG + 16.5% DMSO + 0.67 mol/l sucrose for 25 s. They were then aspirated (0.5 microl) into a plastic micropipette tip (0.36 mm inner diameter), exposed to liquid nitrogen (LN(2)) vapour for 2 min before being placed into a pre-cooled cryotube, which was then closed and plunged into LN(2). Embryos were warmed and diluted using 0.33 mol/l and 0.2 mol/l sucrose. RESULTS: The survival rate for compacted morulae was 73% (22/30) and 82% (27/33) for early blastocysts. CONCLUSIONS: The survival rates of human compacted morulae and early blastocysts after vitrification with this simple technique are similar to those reported in the literature achieved by slow cooling and other vitrification protocols.     

A strategy for rapid cooling of mouse embryos within a double straw to eliminate the risk of contamination during storage in liquid nitrogen

Double packaging, in which an inner straw containing the specimen is inserted into an outer, larger straw (here termed 'straw-in-straw') to prevent the inner straw from coming into direct contact with liquid nitrogen provides a simple strategy for reducing or eliminating the potential contamination risk associated with storage in liquid nitrogen. This approach has in the past been used in conjunction with cryopreservation by slow cooling, but has not previously been tested for use throughout an entire rapid cooling and warming procedure. This study determined whether keeping the straw containing the embryos inside a second protecting container throughout the cryopreservation and storage protocol would compromise embryo viability. We established that a cryoprotectant containing a high polymer concentration (35% dextran or Ficoll) together with 25% ethylene glycol (as the penetrating cryoprotectant) was highly effective for day 2 and day 3 mouse embryos in both single and double straws. The survival and development of all cryopreserved embryos, as assessed both in vitro and in vivo, was not statistically different to their untreated controls. This established that a protein/serum-free cryoprotectant solution supplemented with polymers could provide complete protection of mouse embryos. It also shows, for the first time, that embryos can be cooled by direct immersion in liquid nitrogen and warmed by direct immersion into a waterbath within a double straw arrangement to reduce the likelihood of contamination.     

Azoospermic HIV-1 infected patients wishing to have children: proposed strategy to reduce HIV-1 transmission risk during sperm retrieval and intracytoplasmic sperm injection: Case Report

BACKGROUND: To date, assisted reproductive technology (ART) with sperm washing is offered to serodiscordant couples with an human immunodeficiency virus-1 (HIV-1) infected male partner in order to have a child while reducing the risk of transmission to the woman. However, ART programmes are not possible if the man is azoospermic. We report here the first birth following intracytoplasmic sperm injection (ICSI) using frozen epididymal spermatozoa obtained after surgical sperm retrieval in a HIV-1 infected man with obstructive azoospermia. METHODS; Sperm obtained by micro-epididymal sperm aspiration was frozen after density gradient preparation and tested for HIV-RNA and DNA. ICSI with frozen sperm was performed. RESULTS: A twin pregnancy was obtained following ICSI. Two healthy girls were born. Maternal HIV-1 RNA and HIV-1 serology were negative during pregnancy and at delivery. CONCLUSIONS: This case report demonstrates that ART is possible in azoospermic HIV-1 infected men. On the basis of current knowledge, we propose a strategy to reduce HIV-1 transmission risk during sperm retrieval and ICSI in couples where the man is HIV-1 infected and azoospermic.     

Aseptic technology of vitrification of human pronuclear oocytes using open-pulled straws

BACKGROUND: The aim of this study was to compare the viability of human pronuclear oocytes subjected to vitrification using cooling by direct submerging of open-pulled straws in liquid nitrogen versus vitrification by cooling of open-pulled straws located inside a closed 0.5 ml straw (aseptic system). METHODS: Two- and three-pronuclei stage oocytes (n=114) were cryopreserved in super-open-pulled straws by vitrification in 20% ethylene glycol +20% dimethylsulphoxide (DMSO) + osmotic active and neutral non-permeable cryoprotectants with a four-step exposure in 20, 33, 50 and 100% vitrification solution for 2, 1 and 1 min, and 30-50 s, respectively at room temperature, and plunging into liquid nitrogen. Oocytes of group 1 (n=42) were rapidly cooled at a speed of 20,000 degrees C/min by direct plunging of open-pulled straws into liquid nitrogen. Oocytes of group 2 (n=44) were first located in 0.5 ml straws, which were closed at both sides by metal balls, and then plunged into liquid nitrogen. This method resulted in a cooling speed of 200 degrees C/min. For both groups, oocytes were thawed rapidly at a speed of 20 000 degrees C/min using an identical protocol. Oocytes subsequently were expelled into a graded series of sucrose solutions (1.0, 0.75, 0.5, 0.25 and 0.12 mol/l) at 2.5 min intervals. RESULTS: Oocyte development up to expanded blastocyst stage after in vitro culture was 15% in group 1, 14% in group 2 and 29% in an untreated control group. CONCLUSION: The deposition of human pronuclear oocytes in open-pulled straws which are placed inside a hermetically closed container guarantees a complete isolation of oocytes from liquid nitrogen and avoids potential contamination by pathogenic microorganisms. The combination of direct plunging of this container into liquid nitrogen and rapid warming makes this process as efficient as conventional vitrification.     

A modified cryopreservation method increases the survival of human biopsied cleavage stage embryos

BACKGROUND: The relatively poor survival rate of human biopsied cleavage stage embryos following cryopreservation is a significant obstacle in the application of preimplantation genetic diagnosis (PGD). We have attempted to improve cryosurvival of biopsied embryos by modifying the standard embryo cryopreservation technique. METHODS: Biopsied embryos were cryopreserved in 1.5 mol/l 1,2-propanediol in the presence of an elevated concentration of sucrose (0.2 mol/l) and human serum albumin was replaced by maternal serum (20% vol:vol). An additional initial thawing step in the presence of 0.3 mol/l sucrose was also included. RESULTS: The proportion of biopsied embryos which survived cryopreservation with > or =50% of their blastomeres intact was significantly higher using the modified method (138/185; 75%) than that observed using the standard propanediol method (20/46; 43%; P = 0.022). Total blastomere survival was also significantly increased as a result of the modifications (1010/1513; 67% versus 177/385; 46%; P < 0.001). Six fetal hearts have been detected to date following replacement of biopsied embryos cryopreserved with the modified method. CONCLUSIONS: Survival of human biopsied cleavage stage embryos can be restored to a level similar to that of non-biopsied controls by modification of the cryopreservation procedure. Embryos which have been cryopreserved using the modified method can implant following replacement in utero.     

Ovarian tissue viability following whole ovine ovary cryopreservation: assessing the effects of sphingosine-1-phosphate inclusion

BACKGROUND: Cryopreservation is hypothesized to result in apoptosis, contributing to stromal damage and follicle loss in ovarian tissue. This study investigated tissue viability following whole ovine ovary cryopreservation and examined the effects of the anti-apoptotic agent sphingosine-1-phosphate (S-1-P) on ovarian cryopreservation efficiency. METHODS: Whole ovine ovaries were cryoperfused and subjected to slow-freeze, rapid-thaw cryopreservation before a range of functional viability tests were performed. The effects of 20 micromol(-1) S-1-P, in the cryopreservation media, were then assessed against a control cryopreservation media and non-frozen tissue. RESULTS: Granulosa cell viability (assessed by trypan blue) was not significantly affected, however, Ki67 expression, indicative of cellular proliferation, was reduced following cryopreservation (P< 0.05). Following S-1-P supplementation, granulosa cell viability was not affected by either cryopreservation or S-1-P inclusion. Bromodeoxyuridine uptake, demonstrating DNA synthesis, was seen in both cryopreserved and fresh cortical tissue and the viability stain, 5(6)carboxyfluorescein diacetate succinimidyl ester, showed many viable small follicles. Cryopreservation increased arterial endothelial disruption (P< 0.01), but not internal elastic lamina rupture or venous damage. However, S-1-P supplementation did not improve ovarian or vascular tissue survival. CONCLUSIONS: These results are encouraging for whole ovary cryopreservation, demonstrating maintained cell viability, however, they do not support S-1-P inclusion at this concentration to improve tissue viability following cryopreservation.     

Andrology: Pentoxifylline-supplemented cryoprotectant improves human sperm motility after cryopreservation

In this study, human spermatozoa obtained from donors (n = 15)with normal semen characteristics were cryopreserved in humansperm preservation medium, supplemented with the phosphodiesteraseinhibitor pentoxifylline at concentrations of 0, 1, 3 and 10mM. The effect of pentoxifylline on cryopreserved spermatozoawas determined by monitoring changes in sperm motility and acrosomemorphology by labelling the spermatozoa with fluorescein-conjugatedconcanavalin A lectin. Cryoprotectant supplemented with 1 mMpentoxifylline was found to improve post-thaw progressive motilityfrom 15.3 ± 2.4 (control) to 23.1 ± 3.8% (P <0.01), and total motility from 27.4 ± 3.3 (control) to38.2 ± 3.9% (P < 0.05) without reducing the percentageof spermatozoa with normal acrosomal regions, and so appearsuseful for cryopreservation purposes. The beneficial effectsof 1 mM pentoxifylline on sperm motility were shown to be maintainedpost-thaw over a 6 h time course. Cryoprotectant supplementedwith 3 mM pentoxifylline was found to improve only post-thawprogressive motility, from 15.3 ± 2.4 (control) to 20.7± 3.0% (P < 0.05). However, cryopreservation in thepresence of 10 mM pentoxifylline was found to have a significantly(P < 0.01) detrimental effect on acrosome morphology post-thaw,reducing it from 29.0 ± 2.0 (control) to 21.0 ±2.4% without affecting sperm motility. This suggests that assessmentof the acrosomal region may indicate subtle deleterious effectsof cryoprotectant supplements that cannot be determined frompost-thaw motility assessments alone. These findings differfrom previous studies in that a lower concentration of pentoxifylline(1 mM) was found to be optimal for cryopreservation purposes.     

Vitrification of mouse oocytes using closed pulled straws (CPS) achieves a high survival and preserves good patterns of meiotic spindles, compared with conventional straws, open pulled straws (OPS) and grids.

BACKGROUND: We modified the loading of pulled straws into a new closed system, called closed pulled straws (CPS) for holding oocytes for vitrification. The morphological survival, dynamics of meiotic spindles, and fertilization in vitro of vitrified oocytes using CPS were compared with conventional straws, open pulled straws (OPS), and grids. METHODS: Surviving oocytes were stained for spindles and chromosomes after 1, 2 and 3 h incubations, and compared with controls. The capacity of fertilization and embryonic cleavage were examined in vitro. RESULTS: The survival rates of the CPS (79%) and straw (77%) groups were significantly higher (P < 0.05) than the OPS (63%) and grid (39%) groups. At a 1h incubation, vitrified oocytes of four groups had significantly fewer normal spindles than controls (P < 0.05). The straw group was inferior to the others in spindle morphology (P < 0.05). After a 3 h incubation, recovery of vitrified oocytes with normal spindles was significantly improved in all groups (P < 0.05). The percentages of fertilization and blastocyst formation of vitrified oocytes after a 1 h incubation was significantly lower than controls (P < 0.05), but they were improved after 2 or 3 h incubations (P < 0.05). CONCLUSIONS: Oocytes vitrified using CPS, OPS or grids could lessen spindle injuries and expedite recuperation. The survival using OPS or grids is lower. Sufficient culture time for recovery of meiotic spindle would be imperative for fertilization events of vitrified oocytes. CPS has the advantages of achieving a high survival and preserving good spindles.     

Therapeutic immunization with human immunodeficiency virus type 1 (HIV-1) peptide-loaded dendritic cells is safe and induces immunogenicity in HIV-1-infected individuals

Treatments for human immunodeficiency virus type 1 (HIV-1)-positive individuals that augment HIV-1 suppression and have potential for achieving long-term control of HIV-1 viremia in the absence of antiretroviral therapy (ART) are urgently needed. We therefore conducted a phase I, clinical safety trial of a dendritic cell (DC)-based vaccination strategy as immunotherapy for HIV-1-positive individuals on ART. We studied 18 HIV-1-positive subjects on ART who underwent leukapheresis to obtain peripheral blood mononuclear cells for DC generation from monocytes cultured with cytokines. Mature DC were pulsed with three HIV-1 HLA*A0201 Gag, Env, and Pol peptides and one influenza A virus matrix protein peptide. The vaccine was administered to donors randomized to receive two vaccinations, either intravenously or subcutaneously. The primary end points were safety and tolerability of two doses of peptide-DC vaccine (3 million versus 10 million). Secondary end points included gamma interferon (IFN-γ) enzyme-linked immunospot assay responses and clinical correlates of an immune response to vaccination. Autologous DC-peptide vaccine was safe, well tolerated, and feasible for use in all participants. Adverse events were rare. Although the trial was not powered to assess an immunologic response, a significantly increased frequency of HIV-1 peptide-specific IFN-γ-positive cells was observed 2 weeks following the second vaccine, with three individuals responding to all four peptides. DC vaccination was safe, was feasible, and showed promise of immunogenicity in ART-treated, HIV-1-positive individuals. Additional studies of DC immunization strategies for HIV-1 infection are warranted.     

自精保存在辅助生殖技术中的意义（附74例分析）

目的探讨自精冷冻保存在辅助生殖技术中的意义。方法对2008年9月至2010年11月期间辅助生殖技术前进行自精冷冻保存病史进行回顾性分析,项目包括自精保存人数,自精保存原因,自精来源,精液复苏情况,自存精液利用率等项目。结果共有74名男性行精液冷冻。21例患者为附睾穿刺获取精子,其余53例患者均为准备接受辅助生殖（ART）治疗者：12例少弱精子症患者,担心女方取卵日无法取到可用精子,5例患者女方取卵日丈夫无法到中心留取精液,36例患者因取精困难,担心取卵当日取精障碍。冷冻精液共复苏29例,其中可用17例,使用率约58.6%。结论自精冷冻保存在辅助生殖技术中有重要的作用,可以在一定程度上避免由于男性取精困难造成的辅助生殖的失败,缩短此部分患者的治疗周期,减少治疗费用。     

Human oocyte cryopreservation as an adjunct to IVF-embryo transfer cycles

BACKGROUND: The purpose of this work was to develop methods for successful cryopreservation of human oocytes. METHODS: Two cryopreservation procedures were used. Method 1 involved use of 1.5 mol/l propanediol (PrOH)-0.1 mol/l sucrose with medium containing sodium (Na) as cryoprotectant medium, seeding at -7 degrees C, and stepwise dilution of cryoprotectant post-thaw. Method 2 used Na-depleted media with 1.5 mol/l PrOH-0.2 mol/l sucrose for freezing, seeding at -6 degrees C, and use of high sucrose (0.5 and 0.2 mol/l) for cryoprotectant removal. RESULTS: The first method was used in seven patients, and gave poor (12.3%) survival results and no pregnancies. The second method was used in 15 patients (16 cycles), and yielded good survival and fertilization rates (74.4 and 59% respectively), with four pregnancies and five healthy infants born to 11 women receiving an embryo transfer. CONCLUSIONS: Using Na-depleted media along with other alterations in freezing and thawing procedures, human oocyte cryopreservation can provide excellent survival and pregnancy rates.     

Semen parameters of a semen donor before and after infection with human immunodeficiency virus type 1: case report

Semen samples from a donor who seroconverted for human immunodeficiency virus type 1 (HIV-1) during the period that he was donating at our clinic were stored before and after infection. Semen analysis was done on all of these samples before cryopreservation. Retrospectively, both qualitative and quantitative HIV-1 testing was performed on the cryopreserved semen samples to determine the time of primary HIV-1 infection. After HIV-1 infection, semen volume, sperm motility and the percentage of spermatozoa with normal morphology were reduced compared with the same parameters before HIV-1 infection. HIV-1 RNA was intermittently detectable in semen. HIV-1 infection led to a reduction in semen volume, sperm motility and normal sperm morphology in this donor. However, the clinical significance of these findings is unclear. A longitudinal cohort study on the effects of HIV-1 infection on semen quality is necessary to confirm these findings.     

Diagnostic testicular biopsy and cryopreservation of testicular tissue as an alternative to repeated surgical openings in the treatment of azoospermic men

Between May 1996 and May 1998, 64 azoospermic patients underwent an investigative testicular biopsy combined with the cryopreservation of spermatozoa which were retrieved from a simultaneously examined fresh sample. Testicular tissue cryopreservation was carried out in 43 cases (67%) for late intracytoplasmic sperm injection (ICSI) attempts. In all, 23 couples underwent 26 assisted conception cycles; the fertilization rate was 64% with spermatozoa (139/218, 24 cycles), 40% with round spermatids (2/5, one cycle), and 69% with elongated spermatids (9/13, one cycle). The embryo cleavage rate was 84%. A mean number of 2.7 +/- 0.7 embryos were replaced in 24 patients. In two cases, embryo quality was very poor and they were not transferred. Eight clinical pregnancies resulted (35% per patient and 33% per transferred cycle) with an implantation rate of 14.1%: two patients have already delivered and six are ongoing. In conclusion, the cryopreservation of testicular tissue during the first diagnostic biopsy is an alternative to repeated surgical openings and permits patients to initiate an ovarian stimulation cycle with the certitude of having spermatozoa available. Moreover, since only one straw is routinely used for each ICSI cycle, the frozen tissue remains as a sperm source for multiple attempts.     

Reduced survival after human embryo biopsy and subsequent cryopreservation.

Preimplantation genetic diagnosis (PGD) is performed in couples at risk of genetic disease, so as to avoid transfer of embryos which are affected by a monogenic disease or which carry chromosomal aberrations. As in all in-vitro fertilization (IVF) cycles, supernumerary non-affected good-quality embryos may be available after PGD. These embryos can be cryopreserved. So far, limited data on survival after cryopreservation of biopsied human embryos are available. In this study, human embryos of good morphological quality derived from abnormal fertilization were used to evaluate the influence of the embryo biopsy procedure on survival after cryopreservation. Embryos were allocated to three different groups: control (n = 20), drilling-only (n = 16), and biopsy (n = 29). After freezing and thawing, a significantly lower number of blastomeres was intact in the drilling-only group (46/118, i.e. 39.0%, P < 0.01) and in the embryo biopsy group (46/156, i.e. 29.5%, P < 0.0001) than in the control group (85/151, i.e. 56.3%). This difference was reflected in survival rates of embryos. Fifty-five per cent of the control embryos, 37.5% of the drilling-only group, and 33.3% of the biopsy group had at least 50% of their blastomeres intact. After further in-vitro culture, four blastocysts, three from the drilling-only group and one from the biopsy group, developed from the surviving embryos. From this study it can be concluded that current cryopreservation procedures are less successful when biopsied human embryos are cryopreserved, but that surviving embryos can develop to the blastocyst stage and thus may have the potential to develop to term.