Vitrification of human blastocysts with the Hemi-Straw carrier: application of assisted hatching after thawing.

BACKGROUND: The present study was undertaken to examine the usefulness of both vitrification and assisted hatching (AH) on blastocysts that originate from embryos showing different qualities during their cleavage stage. METHODS: A total of 281 blastocysts were vitrified (93 vitrification-warming cycles) in a mixture of ethylene glycol-dimethylsulphoxide-Ficoll and sucrose using the Hemi-Straw (HS) carrier system. After warming, AH using the partial dissection technique was performed in 36 cycles. RESULTS: After warming and culture for 24 h, a total of 168 blastocysts (60%) was suitable for embryo transfers and a total of 25 ongoing pregnancies (27%) was obtained. Forty-nine transfers of 96 no-AH blastocysts and 36 transfers of 72 AH blastocysts resulted in an implantation rate of 13 and 22% respectively (P < 0.05). The percentage of transfers with at least one hatching blastocyst was significantly higher after application of AH (69 versus 33%) (P < 0.001). In all, 73 and 38% of blastocysts showing respectively optimal and non-optimal embryo development during the early stage were available for transfer (P < 0.001). Consequently, implantation rates of 19 and 6% were obtained after transfers of blastocysts showing respectively optimal and poor embryo development. CONCLUSIONS: Artificial opening of the zona pellucida after warming of vitrified blastocysts significantly improved the rate of transfers with hatched blastocysts and the implantation and pregnancy rates. The percentage of blastocysts that survived the HS vitrification procedure and were available for embryo transfer is related to their previous developmental quality.

Births after vitrification at morula and blastocyst stages: effect of artificial reduction of the blastocoelic cavity before vitrification.

BACKGROUND: In 1996, with the introduction of sequential media, we set up a programme of cryopreservation of supernumerary morulae (day 4) and blastocysts (day 5) using a vitrification procedure. Our results showed that the efficiency of the vitrification method was dependent on the stage of embryo development and was negatively correlated with the expansion of the blastocoele. We postulated that a large blastocoele might disturb cryopreservative potential due to ice crystal formation during the cooling step. We analysed therefore the effectiveness of reducing before vitrification the volume of the blastocoelic cavity. METHOD: Day 4 and day 5 embryos were vitrified in 40% ethylene glycol-18% Ficoll and 0.3 mol/l sucrose before plunging the straws directly into liquid nitrogen. Artificial shrinkage of the blastocyst was achieved after pushing a needle into the blastocoele cavity until it contracted. RESULTS: The survival rate post-thawing of day 4 and intact day 5 embryos was correlated with the volume of the blastocoele. In the control group only 20.3% blastocysts or expanded blastocysts survived as compared with 54.5 and 58.5% with morulae and early blastocyst respectively. After puncturing the blastocoelic cavity, an increase in the survival rate of up to 70.6% was noted. The pregnancy rates were improved after the artificial shrinkage procedure (20.5%) compared with the control intact blastocyst group (4.5%) (not significant). After reduction of the blastocoelic cavity, a significant increase in the implantation rate per vitrified blastocyst was observed (12.0 versus 1.4% P < 0.01). CONCLUSIONS: Our results showed that survival rates in cryopreserved expanded blastocysts could be improved by reducing the fluid content. This was presumably because mechanical damage caused by ice crystal formation was avoided. These observations should be considered when establishing a strategy and a protocol for cryopreservation of day 5 embryos.

Embryo reduction in triplet pregnancies after assisted procreation: a comparative study.

OBJECTIVES: To evaluate pregnancy outcome after selective embryo reduction by transcervical aspiration or transvaginal puncture and intrathoracal injection with potassium chloride (KCl) in triplet pregnancies occurring after assisted procreation and to compare this outcome with that for triplets not undergoing embryo reduction. DESIGN: Retrospective case series. SETTING: In vitro fertilization program of the Centre for Reproductive Medicine of the Dutch-speaking Brussels Free University, Belgium, which is a tertiary referral institution. PATIENTS: Seventy-two patients presenting a triplet pregnancy after assisted procreation. INTERVENTION: Transcervical aspiration embryo reduction at 8 to 9 weeks of pregnancy or transvaginal puncture and intrathoracal injection of KCl at 9 to 10 weeks of pregnancy. MAIN OUTCOME MEASURES: Rate of spontaneous embryo reduction, complications relating to the procedure, pregnancy, and neonatal outcome. RESULTS: The rate of spontaneous reduction was 18%. Among the 14 patients undergoing transcervical aspiration, 3 aborted and 4 lost an additional fetus. The transvaginal puncture technique had a lower complication rate (2/19). Neonatal outcome was improved in pregnancies after selective embryo reduction. After transvaginal puncture, the outcome was comparable with that for twin pregnancies after assisted procreation. CONCLUSIONS: Triplet pregnancies after assisted procreation had a poor neonatal outcome. The outcome was improved after spontaneous reduction. Transcervical aspiration should not be used because of its high rate of early and late complications. Transvaginal puncture had less early complications, but the technique might be associated with prematurity and third trimester fetal death. In triplet pregnancies, embryo reduction decreases the number of babies going home per patient, but the quality of life of the remaining babies is improved.

Analysis of the risk factors with regard to the occurrence of ectopic pregnancy after medically assisted procreation.

In a retrospective study (1985-1989) based on data from the Centre for Reproductive Medicine in Brussels, a total of 23 ectopic pregnancies (2.24%) occurred after 3800 embryo, zygote or gamete transfers. This number was low compared with the data published elsewhere. Tubal damage was a major risk factor towards developing an ectopic pregnancy after in-vitro fertilization and embryo transfer. The number of ectopic pregnancies after the association of clomiphene citrate and human menopausal gonadotrophin (HMG) was significantly higher in patients with tubal (7.8%) and non-tubal indications (2.1%) compared with those stimulated with gonadotrophin-releasing hormone (GnRH) and HMG (2.18% and 0.84%, respectively). The number of replaced embryos was not associated with the rate of ectopic pregnancy and neither did transfer technique (intra-uterine or intra-Fallopian transfer) influence the ectopic pregnancy rate.

Laparoscopic removal of benign mature teratoma.

Laparoscopic removal of benign mature teratoma is safe and effective in pre-menopausal patients. Fourteen patients underwent laparoscopic removal of such tumours. There were no operative complications. Post-operative hospitalization was short (average: 2 days). All patients undergoing a second-look laparoscopy had mild adhesions around the operated ovary. Two spontaneous and one in-vitro fertilization pregnancy occurred. If a teratoma is diagnosed during laparoscopy or oocyte retrieval, it should be promptly removed as malignant transformation is not excluded. In this case, the embryos should be frozen for later transfer.

The effect of gonadotrophin-releasing hormone (GnRH) agonist in the follicular phase on in-vitro fertilization outcome in normo-ovulatory women.

Sixty-three normo-ovulatory infertile women were randomly divided into two groups. All women were first desensitized with the gonadotrophin-releasing hormone agonist (GnRHa), buserelin. Thereafter, ovarian stimulation with human menopausal gonadotrophins (HMG) was started in both groups but in group A the GnRHa was stopped on the same day. In group B, the GnRHa was continued during HMG treatment until the ovulatory human chorionic gonadotrophin stimulus was given. Premature luteinization was not observed in either group, although the preovulatory basal luteinizing hormone (LH) secretion was significantly higher in group A. An equal number of embryos of comparable quality was transferred in both groups and the pregnancy outcome was similar. However, the supernumerary embryos of group A were of a lower morphological quality and survived the cryopreservation process less well. We concluded that the continuous administration of a GnRH agonist during HMG treatment resulted in better quality of supernumerary embryos.

Zygote intrafallopian transfer or in vitro fertilization and embryo transfer for the treatment of male-factor infertility: a prospective randomized trial.

OBJECTIVE: To compare zygote intrafallopian transfer (ZIFT) and in vitro fertilization and embryo transfer (IVF-ET) as treatments of male-factor infertility. DESIGN: Patients were prospectively randomized to ZIFT or IVF-ET. SETTING: In vitro fertilization program of the Centre for Reproductive Medicine of the Dutch-speaking Brussels Free University, Belgium, which is a tertiary referral institution. PATIENTS: One hundred fifty-seven couples were enrolled in the study. Inclusion criteria allowed only first trials of couples with long-standing infertility caused by a male factor. Female factors were excluded. INTERVENTIONS: In ZIFT, up to three fertilized oocytes were transferred into one single patient fallopian tube by means of laparoscopy 18 hours after insemination. In IVF-ET, cleaving embryos were replaced into the uterine cavity about 48 hours after insemination. MAIN OUTCOME MEASURES: Fertilization and transfer rates, implantation and pregnancy rates, pregnancy outcome, and cost per procedure were evaluated. RESULTS: Implantation rates of 12.3% and 10% per replaced conceptus were achieved for ZIFT and IVF-ET, respectively. CONCLUSIONS: This study demonstrates no therapeutic advantage of ZIFT over IVF-ET in male-factor infertility in terms of reproductive outcome or economic benefit.

The relationship between embryo quality and the occurrence of multiple pregnancies.

OBJECTIVE: To study the relationship between the quality of the transferred embryos and the occurrence of multiple pregnancies. Embryo quality was defined by the cleavage rate and by morphological parameters such as blastomere size and the presence or absence of anucleate fragments. DESIGN: A retrospective analysis of 1,915 consecutive transfers of fresh embryos between January 1986 and December 1989. SETTING: All the embryo transfers (ETs) were performed in patients from the in vitro fertilization program of the Center for Reproductive Medicine, Vrije Universiteit Brussel, Brussels, Belgium. RESULTS: We confirmed the relationship between the number of embryos transferred and the pregnancy rate (PR): 11.9% of the single, 19.0% of the double, and 34.1% of the triple ETs were successful. Thirty-one percent of these triple embryo replacements resulted in a multiple gestation. At the time of transfer (44 to 48 hours after insemination), we observed that embryos that had undergone at least two mitotic divisions implanted better than two-cell embryos of comparable morphological appearance (implantation rate per transferred embryo: 21.3% versus 12.3%, P less than 0.001) and that heavily fragmented embryos did not implant as well as embryos without or with fewer anucleate fragments (1.5% versus 14.1%, P less than 0.001). The PR, implantation rate, and the incidence of multiple pregnancies increased significantly with the number of good quality embryos that were transferred. CONCLUSIONS: Our study indicated that embryo quality based on morphological observations could predict the occurrence of multiple pregnancies.

Comparison of in-vitro fertilization in male and tubal infertility: a 3 year survey.

The in-vitro fertilization and embryo transfer (IVF-ET) procedure was compared in 175 couples with male infertility and 480 couples with tubal infertility. In cases of male infertility, more oocytes were recovered but fewer oocytes were fertilized. Although the cleavage rate was decreased, no difference in embryo quality was found. In male infertility, fewer transfers were performed and the average number of embryos per transfer was lower. The total pregnancy rate was also lower per cycle (12.8 versus 22.9%), but not per transfer (25.4 versus 25.7%). The further obstetrical outcome was similar in both groups. We conclude that male infertility can be treated by IVF-ET but results are still disappointing when compared to a control group with normal spermatozoa.

The influence of inadvertent intranasal buserelin administration in early pregnancy.

Buserelin was inadvertently administered during 13 early pregnancies in 12 women with long-standing infertility, who had started the GnRH-agonist for gonadotrophin desensitization prior to ovarian stimulation for IVF. Six women delivered a healthy child and one pregnancy continues uneventfully. Three patients aborted before the sixth week and three women with tubal disease had an ectopic pregnancy. Corpus luteum function was normal in 11 of the 13 pregnancies. Although no evidence of embryotoxic effects of buserelin was observed, barrier contraceptive methods should be advised during the first days of its administration.

The incidence of multiple pregnancy after in vitro fertilization and embryo transfer, gamete, or zygote intrafallopian transfer.

This retrospective study concerns the incidence of multiple pregnancy after the replacement of three conceptus using different techniques of assisted reproduction. During a 2-year period, 713 in vitro fertilization-embryo transfers (IVF-ETs) with three embryos, 190 gamete intrafallopian transfers (GIFT) with three oocytes, and 161 zygote intrafallopian transfers (ZIFT) with three zygotes were performed. Although we observed significant differences in implantation and pregnancy rates (PRs), the three techniques resulted in high multiple PR. At 20 weeks, 16% of GIFT pregnancies, 27% of ZIFT pregnancies, and 32% of IVF-ET pregnancies were multiple. Therefore we recommend to limit the number of conceptus transferred to a maximum of three in all cases.

In vitro fertilization techniques with frozen-thawed sperm: a method for preserving the progenitive potential of Hodgkin patients.

The banked sperm of 5 of 13 Hodgkin patients was thawed for assisted reproduction. The post-thaw sperm characteristics were extremely impaired. Nevertheless, in 80% of all trials fertilization in vitro was obtained and conceptus could be transferred. All spouses became pregnant and seven healthy infants were born. One could recommend every Hodgkin patient to bank his sperm before chemotherapy. In vitro fertilization techniques with this frozen-thawed sperm reveal promising results in comparison with artificial insemination to maintain the reproductive capacity of male Hodgkin patients.

Short-term use of buserelin in combination with human menopausal gonadotrophins for ovarian stimulation for in-vitro fertilization in endocrinologically normal women.

Ten endocrinologically normal women were injected subcutaneously with 500 micrograms D-Ser(TBU)6-EA10-LHRH (buserelin) on days 3,4 and 5 after the start of the menses. Two types of response were observed. Five women (group A) responded promptly and had a mean number of 13.4 oocytes retreived after 11.4 days of stimulation. In the second group (B), two to three times more HMG was needed to obtain a mean number of 7.3 oocytes after 17.2 days of stimulation. The response upon stimulation could be predicted by the serum gonadotrophin output on days 4 and 5 of the cycle. One woman from group B had a premature LH rise on day 16 and luteinization; her cycle was abandoned. In the four other patients of group B, serum and urinary LH concentrations showed that pituitary gonadotrophin secretion had recovered before the ovulatory stimulus, without signs of premature luteinization. Two women in each group became pregnant, one of whom aborted. This short-term GnRH agonist treatment could be an alternative method for ovarian stimulation, although it did not totally prevent the occurrence of an endogenous LH surge.

Effect of transuterine puncture during transvaginal oocyte retrieval on the results of human in-vitro fertilization.

In many in-vitro fertilization (IVF) programmes, the transvaginal approach is now the most widely used method for ultrasound-guided oocyte retrieval. Sometimes, however, the position of the ovaries is such that it might not be possible to aspirate the follicle transvaginally without puncturing the uterine wall. Between May and November 1988, we performed 518 transvaginal, ultrasound-guided oocyte retrievals for IVF or zygote intra-Fallopian transfer (ZIFT). During this period, 22 transuterine punctures (4.2%) were necessary to reach one of the ovaries. To investigate the effect of uterine laceration on the results of IVF/ZIFT treatment, the outcome of treatment was compared between patients who had a transuterine puncture and a control group of patients treated by IVF or ZIFT during the same period without transuterine puncture. Seven of 16 patients who had a transuterine puncture became pregnant after IVF, compared to 84 pregnancies out of 347 cases (24.2%) in the control group. After ZIFT, two pregnancies (2/6) were achieved in patients with uterine laceration, compared to 46 pregnancies out of 149 procedures (30.8%) in the control group. These differences were not statistically significant. Transuterine puncture during transvaginal oocyte retrieval does not seem to have an adverse effect on the results of human IVF and ZIFT treatment.