Cryopreservation of biopsied embryos at the blastocyst stage.

BACKGROUND: The availability of an efficient cryopreservation program is especially important in the case of embryos that have undergone blastomere biopsy for PGD. Unfortunately, the freezing/thawing of biopsied embryos has given disappointing results when performed at the cleavage stage. In this study, embryos diagnosed as normal after PGD were grown to the blastocyst stage, frozen and thawed for successive frozen embryo transfer. METHODS: A total of 34 patients performed a thawing cycle in which 47 blastocysts were thawed. The cryopreservation solutions were based on HEPES-buffered medium supplemented with human serum albumin (HSA), sucrose and 1,2-propanediol. The same protocol was applied to embryos from 88 IVF/ICSI patients, which underwent 92 thawing cycles with 150 thawed blastocysts. RESULTS: The survival rate was similar in the two groups (53% after PGD and 58% in IVF/ICSI cycles), as well as the cumulative pregnancy rate per patient (59% after PGD versus 47% in IVF/ICSI cycles), despite a higher maternal age and a lower proportion of embryos available for transfer or cryopreservation in the PGD group. CONCLUSIONS: Neither the survival rate nor the subsequent development and chances of implantation, differed between embryos frozen at the blastocyst stage following biopsy and those frozen intact.

Vero cell effect on in-vitro human blastocyst development: preliminary results.

Asynchrony between embryo and uterine environment is one of the major limits in human in-vitro fertilization (IVF). A culture system which could prolong culture time and increase embryonic cleavage rate and viability would improve success rates. Using Vero cells, an in-vitro co-culture system was developed to investigate and promote human embryo development. Vero cells provide good support for human early embryos up to the blastocyst stage. When fertilized embryos were co-cultured, 68% of them reached the blastocyst stage. Pregnancy rate was 50% per transfer in patients with several previous failures of implantation. A significant increase in clinical pregnancy rate was also demonstrated when zygotes were maintained on Vero cell monolayer for only 24 h. The beneficial effect of the feeder layer may be through the release of embryotrophic factors and the detoxification of the culture medium by the cells. Co-culture is a new concept in assisted reproduction.

What is the most idoneous developmental stage for embryo-freezing?

Mammalian preimplantation embryos can be cryopreserved by a variety of methods involving different cryoprotectants. In the present study, we ultrarapidly froze 299 excess embryos at various early cleavage stages (zygotes, 2-cell embryos, 4-cell embryos and > 5-cell ones) after a brief exposure to high concentrations of dimethyl sulfoxide (DMSO; 3.5 M) and 0.25M sucrose. One-hundred seventeen of them were thawed in a warm water bath. Thirtyone embryos were completely destroyed after thawing but only 6% of them were originally at the pronuclear stage. Regarding the morphological aspects, 94% of pronucleate embryos were intact whereas just 12% of > 5-cell embryos showed more than 50% intact blastomeres after thawing. We froze also 60 embryos at the blastocyst stage by a slow freezing procedure. Eighteen blastocysts were thawed and 12 of them fully re-expanded were transferred in four patients.

Assisted fertilization with epididymal spermatozoa.

In vitro fertilization is used for several years as a technique for resolving infertility problems due to moderate or severe oligospermia. More recently, techniques of micro-insemination of oocytes have also become available for cases of extremely severe oligospermia which cannot be resolved by classical I.V.F. Nevertheless, although these particular techniques have already led to results which have gone far beyond initial hopes, they are not able to resolve all cases of male sterility. There are indeed many situations of excretory azoospermia associated with normal spermatogenesis; the spermatozoa remain trapped in a more or less extensive part of the epididymis because its passage is blocked, either because of post-infectious sclerosis, or of agenesis of a variably extensive area of the Wolffian duct. Post-inflammatory occlusions can be treated by micro-surgery, whereas in cases of agenesis, attempts to collect spermatozoa by means of an artificial spermatocele have led to far too many failures, and this technique has now been abandoned, in spite of some successful pregnancies. The extraordinary development of in vitro fertilization techniques has led to the logical idea that it might be possible to collect epididymal spermatozoa for oocyte fertilization.

Successful pregnancy in a pure gonadal dysgenesis with karyotype 46,XY patient (Swyer's syndrome) following oocyte donation and hormonal treatment.

A pregnancy obtained in a patient with 46XY gonadal dysgenesis (Swyer's syndrome) after ovum donation and IVF/ET is described. The efficacy of this technique in overcoming particular cases of otherwise irreversible infertility is discussed.

Successful gamete intrafallopian transfer following failed artificial insemination by donor: evidence for a defect in gamete transport?

Gamete intrafallopian transfer (GIFT) was offered as an alternative treatment to 48 women who failed to conceive after artificial insemination with donor semen (AID) in numerous attempts (9 to 24 cycles). The evaluation of these women showed no major cause of infertility as evidenced by normal endocrine, cervical, uterine, and tubal factor studies. Their partners were either azoospermic or severely oligoasthenospermic. During the GIFT cycle, follicular development was induced with (1) clomiphene citrate (days 3 to 7) plus human menopausal gonadotropins (hMG) from day 6 on or (2) human follicle-stimulating hormone (days 3 to 4) plus hMG (day 5 on), until ultrasound revealed 2 follicles 16 mm and serum estradiol (E2) was greater than 700 pg/ml. Human chorionic gonadotropin (hCG) 10,000 IU was administered, and 36 hours later follicular aspiration was performed. One to three oocytes and 100,000 motile sperm were transferred to each fallopian tube through the fimbria via laparoscopy or minilaparotomy. Twenty-seven clinical pregnancies were achieved (56%) per GIFT cycle. Eight miscarriages occurred during the first trimester (29% of all pregnancies), whereas no ectopic pregnancies were observed. These data conclusively show the value of the GIFT procedure in the treatment of cases with failed AID.

Oocyte donation and gamete intrafallopian transfer in premature ovarian failure.

Gamete intrafallopian transfer (GIFT) was performed in eight patients with premature ovarian failure (POF), using donated oocytes. The steroid replacement protocol consisted of the administration of increasing dosages of 17 beta-estradiol (E2) and progesterone (P). Hormonal replacement was maintained until day 100 of gestation. All patients underwent an evaluation cycle in which serum levels of E2 and P were monitored and an endometrial biopsy was performed on day 21 or 22. All cases of GIFT were performed between days 12 and 15. Six clinical pregnancies were achieved in eight cycles (75% success rate). Three patients delivered and three are in their second or third trimester. No ectopics or miscarriages occurred. These results offer a promising approach for the establishment of fertility in agonadal patients.

Isolation of a histamine releasing factor from human embryo culture medium after in-vitro fertilization.

In-vitro fertilization (IVF) of human oocytes in our laboratories gave a percentage pregnancy rate per transfer close to 20% during 1985. Embryos were grown until the two-four cell stage and then transferred to the maternal uterus. The media from these embryo cultures were collected and subjected to chromatography on heparin-Sepharose affinity columns. The bound protein fraction contained a factor capable of inducing histamine release from sensitized basophils. The effect of this embryo-derived histamine-releasing factor (EHRF) was to induce a maximum 56 +/- 7% release of the total histamine available. This value varied between 20 and 60%, resulting from 10-30 micrograms/ml of EHRF. Since the histamine release assay performed with basophils from non-atopic donors gave no positive results, we conclude that the release was not due to a cytotoxic mechanism. This was also supported by the absence of histamine release when the assay was performed at 0 degree C, or in the presence of 2 mM EDTA, suggesting that release was dependent on an immunological interaction between EHRF and some receptor on the basophils. The immunosuppressive role of histamine is well known, and a model involving EHRF and histamine is suggested here to explain the mechanism mounted by the embryo to escape maternal immune rejection.

Gamete intrafallopian transfer in the treatment of infertility: the first series at the University of Palermo.

Twenty-six couples with unexplained infertility (UI), nine women with repeated failures of artificial insemination with donor semen (AID), three women with mild endometriosis, three with periadnexal adhesions, one with hostile (not immunologic) cervical mucus, and one couple in which the male partner was affected by asthenospermia were treated by the gamete intrafallopian transfer (GIFT) technique. Three different protocols for controlled ovarian hyperstimulation were used, and an adequate follicular growth and oocyte maturation were achieved in all cases. Seventeen pregnancies were obtained, for a global pregnancy rate of 38.6%. Two pregnancies (11.7%) ended in clinical abortions, and one (5.8%) was a tubal pregnancy. Of the ongoing pregnancies, one is twin and two are triplets. Seven pregnancies (six ongoing, one abortion) were obtained in the UI group (26%), six (all ongoing) in the failed AID group (66.6%), two continuing pregnancies in the three patients with endometriosis (66.6%); the tubal pregnancy and one clinical abortion occurred in the group with adnexal adhesions. No pregnancies were obtained in the patient with hostile cervical mucus and in the couple with infertility presumably due to poor semen. These encouraging results and the simplicity of the technique suggest that GIFT could be an effective approach that could be programmed during a well-timed laparoscopy where persistent infertility exists in association with apparently normal fallopian tubes.

One year's experience with the GIFT method.

From January 1985 to December 1985 we have performed 83 GIFTS for various indications such as unexplained infertility, immunological problems, endometriosis, male factor, fimbrial pathology. In 82 women out of 83, oocytes and sperm have been transferred and 31 pregnancies (37.8%) have been obtained. Twelve women have delivered 17 babies; 11 pregnancies are ongoing, 7 have been interrupted by an abortion and 1 by a tubal pregnancy. The present and future roles of GIFT in the treatment of infertility is discussed.

Gamete intra-fallopian transfer (GIFT): a new technique for the treatment of unexplained infertility.

The Gamete Intrafallopian Transfer (GIFT) was carried out in 12 couples with unexplained infertility, in 2 cases with infertility associated with mild endometriosis, in 1 case of hostile mucus and 3 cases in which phymosis of the ampulla and/or periadnexal adhesions were previously identified. In 7 couples GIFT was carried out after several AID cycles for husband azoospermia or severe oligospermia. Three different protocols were used for the controlled ovarian hyperstimulation. In all cases an adequate follicular growth was obtained and mature oocytes were recovered. At first attempt 12 ongoing pregnancies (1 triplet, 1 twin) were obtained with a present pregnancy rate of 48%.

In vitro fertilization as an out-patient procedure.

A new, non invasive method for IVF/ET is discussed. Twenty infertile patients were involved in an outpatient procedure; less risk for them, because of local anaesthesia and low costs for the clinic are the most remarkable advantages of this procedure. Four normal ongoing pregnancies followed this treatment.

Monitoring of follicular growth for I.V.F.-E.T. programme: the value of E2 assay and echography.

The possibility of a correlation between biological (E2) and morphological (U.S.) parameters, in view of obtaining a correct prevision of follicular maturation and ovulation is discussed. In this study normal patients with a tubal sterility were treated with clomiphene citrate and clomiphene plus H.M.G. The statistical analysis was performed by means of the linear correlation (r) and the relative test of significance (Student T.). The data obtained in this study show that notwithstanding that the rising plasma concentration of E2, which precedes ovulation, is the first and more constant marker of follicular maturation, because of its wide range of variability its usefulness in prediction of ovulation is nearly limited to spontaneous cycles. In fact in stimulated cycles the rising plasma-E2 concentration is rather an independent variable regarding the growth of single follicles (F.D.).

Modulation of lymphocyte response by hormones.

The present study was carried out to investigate the influence of inhibitor factors on the immunological response of the mother. Using a FIVET program (in vitro fertilization and embryo-transfer) we were able to detect in vitro the modulation of lymphocyte response by hormones, utilizing subjects receiving hormone therapy. A new assay has been used to study the antibody response. The lymphocytes were adhered during the culture to the plastic surface of microplates, and the modulation of the expression of the surface IgG was detected with an E.L.I.S.A. assay in the same culture plate. Many hormones were tested in vitro using this assay. The addition to the lymphocyte cultures of hCG and oestradiol give a variable effect. This effect is concentration dependent and whilst in all instances the higher dosages were inhibitory, in same subjects the lower dosages had a stimulatory effect, but if the subjects were treated in vivo with hCG, then an inhibitory effect was observed in vitro even using very low concentrations of hormones. This could suggest that these hormones have an inhibitor effect on the lymphocyte response, inhibition that was enhanced when the lymphocytes were "primed" in vivo.