Hilton I. Kort

The efficacy and safety of human oocyte vitrification.

Vitrification is now a widely applied and highly successful approach for cryopreservation in reproductive biology. Rapidly increasing data prove that it is also a highly efficient technique for low-temperature storage of human oocytes. The latest approaches with appropriately selected cryoprotectants, tools and techniques, and properly adjusted parameters allow close to 100% morphological survival rates, and in vitro embryo development, as well pregnancy and implantation rates, comparable with those achieved with fresh oocytes. With standardization of the technique and elimination of biosafety problems by preserving all the positive features, vitrification may become a common part of the everyday routine in a human embryo laboratory, and it may offer a solution for various medical and social situations as well as for simple logistic problems commonly occurring in assisted reproduction.

Increased Pregnancy Rate with Oil-Soluble Hysterosalpingography Dye*

The diagnostic value of hysterosalpingography remains unquestioned. Previous studies have suggested that patients who underwent hysterosalpingography with an oil-soluble contract medium (OSCM) rather than a water-soluble contrast medium (WSCM) had higher subsequent fertility rates. This study evaluated subsequent fertility rates in 339 patients who underwent hysterosalpingography in which OSCM or WSCM was used. All forms of infertility were included and over-all, pregnancy occurred in the WSCM group in 21 of 162 patients (13%), in the OSCM group in 51 of 177 patients (29%); these rates were significantly different (P less than 0.001), with the unexplained infertility group and the male factor infertility group showing the most significant difference. Adverse effects of OSCM hysterosalpingography were not corroborated. For this reason it is suggested that initially the patient undergo hysterosalpingography with WSCM. Once patency has been established, 3 ml of OSCM should be injected as a therapeutic modality to increase subsequent fertility.

Electroejaculation in combination with in vitro fertilization and gamete micromanipulation for treatment of anejaculatory male infertility

Objective: Failure to ejaculate may be overcome by use of electroejaculation. However, such semen samples are often unsuitable for therapies like intrauterine insemination. The combination of electroejaculation with in vitro fertilization, including gamete micromanipulation, should improve chances of fertilization and pregnancy in such cases. Study Design: Within a private infertility clinic electroejaculation in combination with intrauterine insemination was carried out in 18 cycles (10 couples). Four couples went on to receive therapy by electroejaculation plus in vitro fertilization, along with six other couples (15 cycles total) with semen too poor for intrauterine insemination. Results: One term pregnancy arose in the electroejaculation-intrauterine insemination group, and one term pregnancy plus one continuing pregnancy arose from two couples (three cycles) who underwent in vitro fertilization with conventional insemination after electroejaculation. Six couples (nine cycles) had embryos arising only from gamete micromanipulation transferred, and this yielded two term pregnancies, one spontaneous abortion, and a biochemical pregnancy. Two couples (three cycles) failed to achieve fertilization even with micromanipulation; however, donor-inseminated eggs gave rise to two term pregnancies and one continuing pregnancy in these patients. Conclusions: This report confirms the feasibility of in vitro fertilization in conjunction with electroejaculation and extends the therapy to incorporate gamete micromanipulation.

Two successful pregnancies obtained following oocyte vitrification and embryo re-vitrification

Recent clinical reports not only show that cryopreserved embryos can be successfully used for human fertility treatment, but also that cryopreserved oocytes may be used successfully as an adjunct to human assisted reproductive technologies. Vitrification is known to establish a glass-like solid state during the cooling process. The high concentration of cryoprotectants and an extremely rapid rate of cooling are responsible for the formation of the solid state, and also prevent formation of intracellular ice crystals. Hence, in theory, vitrification should minimize cryo-injuries, and therefore has great promise for oocyte and embryo cryopreservation. This article describes two pregnancies from vitrified-warmed blastocysts obtained after intracytoplasmic sperm injection fertilization of vitrified-warmed oocytes. Vitrification was employed to cryopreserve the oocytes and the subsequent blastocysts. The results present the intriguing implication that vitrification may serve as an efficient method for clinical oocyte cryopreservation and embryo re-cryopreservation.

Human oocyte vitrification: in-vivo and in-vitro maturation outcomes

This study aimed to evaluate oocyte vitrification efficiency using in-vivo matured (IVO) versus rescued in-vitro matured (IVM) oocytes. The results show that oocyte survival (85% versus 81%), fertilization (86% versus 76%) and cleavage rate (98% versus 89%) was not significantly different in IVO oocytes compared with rescued IVM sibling oocytes. The fertilized oocytes from IVO and IVM groups were cultured to blastocyst stage; however, embryo development was significantly reduced in the rescued IVM group (72% versus 15%). Embryo transfer was only performed with the embryos derived from IVO oocytes on day 5; 42 blastocysts were transferred to 18 recipients; 16 of 18 recipients had positive beta-human chorionic gonadotrophin and a total of 26 fetal cardiac activities were detected in 15 recipients (implantation: 26/42, 61.9%). Ten of the 15 recipients have delivered 19 healthy babies, and the other five pregnancies are still ongoing. These data indicate that the combination of oocyte vitrification and rescued IVM not only yield a new strategy to extend the pool of total fertilizable oocytes, but also demonstrate that the efficiency of vitrified/warmed oocytes can be comparable to fresh oocytes with regard to clinical outcomes.

Microsurgical technique in the management of tubal ectopic pregnancy.

Microsurgical techniques employing fine instruments and suture, microelectrocautery and continuous lavage, and careful handling of tissue have been applied to a variety of gynecologic procedures in which the microscope itself is not warranted. The thesis underlying this practice is straightforward: if successful outcome in chronic tubal disease is enhanced by these procedures, then they may have a beneficial role in acute tubal disease. To test this premise, in the past 2 years nine patients with tubal ectopic pregnancies have been managed by microsurgical techniques at Yale-New Haven Hospital. Five of the ectopic pregnancies were unruptured and four were ruptured. The products of conception were removed either by meticulous debridement or linear antimesenteric salpingostomy in association with the microsurgical methods noted above. Tubal healing was by secondary intention. All patients were treated with prophylactic antibiotics and intra-abdominal low molecular weight dextran. Four months postoperatively, all tubes operated upon were patent by salpingography. Subsequently, three of the five women with unruptured pregnancies and two of the four with ruptured pregnancies achieved intrauterine pregnancies. However, one has spontaneously aborted. According to these preliminary results, microsurgical techniques may be beneficially applied in the treatment of selected cases of ectopic pregnancy.

Impact of phase transition on the mouse oocyte spindle during vitrification

During vitrification, the glass-like solidification is the phase-transition process from liquid to solid. Phase transition is one of the major factors suspected to affect the physiology of the oocyte, such as the structure of the meiotic spindle. Therefore, it is very important to investigate the systematic and morphological alterations of the metaphase-II spindle and chromosome arrangement during complete course of a vitrification and warming process. B6D2F1 (C57BL/6 X DBA/2) mouse oocytes were cryopreserved by minimum volume cooling (MVC) method of vitrification in a solution with 15% ethylene glycol, 15% dimethylsulphoxide and 0.5 mol/l sucrose. To examine the spindle, oocytes were fixed before, during and after vitrification and were analysed by immunocytochemistry and confocal microscopy. It was shown that spindles in all oocytes could be maintained through the vitrification and warming process, even though they were exposed to extreme temperature and two rounds of phase transition. According to the sequential observations, chromosome alignment was maintained throughout the complete course of vitrification, warming and post-warming stage. The impact of phase transition was barely detectable when the oocyte was exposed to the vitrification and warming process. The oocyte spindle was able to recover immediately after warming.

Effect of coculture on subsequent survival and implantation of cryopreserved human embryos

PurposeThis retrospective analysis was designed to assess the performance of human embryos following cryopreservation based on whether they were originally developed in standard culture medium (65 cycles, 223 embryos) or cocultured on partial monolayers of bovine oviductal epithelial cells (63 cycles, 198 embryos). Embryo cryosurvival and implantation were compared between the study group and the contemporaneously matched controls.ResultsDuring a 2-year period when no factors of the cryopreservation program were altered, 63 transfers of 159 surviving thawed control cleavage-stage embryos (71.3% survival) that were 54% intact gave rise to 11 viable pregnancies (17.5%/ET), to yield an implantation rate of 6.9% per embryo. Sixty-three transfers of 147 thawed cocultured embryos (74.2% survival) that were 61% intact gave rise to 17 viable pregnancies (27%/ET), which gave an implantation rate of 13.6% per embryo that was significantly higher than the control group (P< 0.05).ConclusionCoculture of embryos prior to cryopreservation does not appear to improve cryosurvival; however, it does improve implantation postthaw compared with embryos following standard culture prior to cryopreservation.

Comparison of ICSI and conventional IVF in patients with increased oocyte immaturity

Using sibling oocytes, the objective of this study was to compare the intracytoplasmic sperm injection (ICSI) fertilization rates to those achieved with conventional IVF in patients with high rates of oocyte immaturity. This study was observational in nature, and included 91 patients who were treated using split insemination techniques. The fertilization rates for the ICSI group and the IVF group were 41.1 +/- 15.0% and 53.2 +/- 19.8%, respectively (P <: 0.0001). There was no significant difference in day-3 embryo quality between the two groups. There was a significantly higher number of embryos frozen in the IVF group than in the ICSI group: 357 (84.8%) and 297 (76.7%), respectively (P = 0.037). Furthermore, the number of embryos either transferred or frozen was significantly higher in the IVF group than the ICSI group: 459 of 1173 (39.1%) and 385 of 1268 (30.4%), respectively (P < 0.0001). These data indicate that conventional IVF results in a higher fertilization rate than ICSI. Furthermore, IVF provided more embryos available for transfer or cryopreservation when compared with ICSI, thereby optimizing the patients cycle.

Poor implantation of cryopreserved reinsemination-fertilized human embryos

OBJECTIVE To investigate whether a poor rate of implantation after in vitro fertilization (IVF) was due to poor embryonic/endometrial synchrony during the original IVF cycle, we have cryopreserved reinseminated-fertilized embryos for later more synchronous replacement after thawing. The chance of implantation of fresh reinseminated fertilized human oocytes is approximately one tenth that of timely fertilized embryos. STUDY DESIGN AND DATA: Retrospective study of 35 original oocyte collections in which initial normal fertilization was 47.3% (129/273 oocytes), with 49.6% fertilization (67/135) upon reinsemination. Of these, 70 initially fertilized and 67 reinsemination-fertilized embryos were cryopreserved, and 50 initially fertilized and all 67 reinsemination-fertilized embryos were subsequently thawed with 72% and 63% cryosurvival, respectively, (not significant). SETTING Private infertility clinic. RESULTS In 11 cycles, 23 thawed initially fertilized embryos (group A) were replaced with a 21.7% implantation rate per embryo; in 10 cycles, 13 initially fertilized and 12 reinsemination-fertilized embryos (group B) were replaced together with an 8% implantation rate; finally, in 16 cycles, 30 reinsemination-fertilized embryos (group C) were replaced with a 3.3% implantation rate (group A versus group C: P = 0.076). Comparison of clinical pregnancies between these groups was significantly different (6/11 versus 1/16; P = 0.0427). CONCLUSION Reinsemination-fertilized embryos survive freezing as well as initially fertilized embryos, but better embryonic/endometrial synchrony does not improve chances of their implantation.