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.

Intracytoplasmic injection of spermatids retrieved from testicular tissue: influence of testicular pathology, type of selected spermatids and oocyte activation.

Spermatid microinjection into oocytes has proven to be a successful assisted reproduction procedure in the animal model and in the human species, since in the latter a few full-term pregnancies were actually obtained. Patients entering our spermatid injection study included those with a total absence of spermatozoa in the testicular tissue notwithstanding previous positive biopsies (n = 29): an obstructive problem (n = 3), secretory azoospermia (n = 26), and those with total arrest at the spermatogenesis level in previous explorative biopsies (n = 15). In the latter group, absence of spermatids was recorded in four cases. Mature, elongated, elongating and round spermatids (ROS) were injected in respectively 3, 2, 3, and 32 attempts. A total of 260 metaphase II oocytes were injected with ROS, 36 oocytes with spermatids at other stages of maturity. The rates of oocytes showing two pronuclei (2PN) and two polar bodies reached 22% and 64% respectively after injection of round or elongated-mature spermatids. The fertilization rate after ROS injection was influenced by the percentage of spermatozoa observed in a previous biopsy. Patients with a positive preliminary biopsy had significantly more 2PN (33%) when compared to those with a severe spermatogenic dysfunction and in whom no spermatozoa were found (only 11%) (P < 0.05). Incubation of oocytes in calcium ionophore after ROS injection had a positive effect on the rate of 2PN formation (36 versus 16%). Ninety per cent of all the normally fertilized oocytes cleaved. The percentage of grade A and B embryos depended on the type of injected cells: 12% after ROS and 30% with the other types of haploid cells. A total of 39 transfers resulted in five pregnancies: three full term with healthy babies delivered (one after ROS injection, and two after injection of an elongating and a mature spermatid), one 4 months ongoing (after elongating spermatid injection) and one miscarriage at 4 weeks (after elongated cell injection). Compared to our conventional intracytoplasmic sperm injection-testicular sperm extraction (ICSI-TESE) programme, the implantation rate after ROS injection was very low (5.5 versus 10.5%).

[Medically assisted reproduction with immature spermatozoa. Clinical examination and surgical technics]. / La procréation médicalement assistée avec spermatozoïdes immatures. Examens cliniques et techniques chirurgicales.

The authors report their experience with the use of spermatids in TESE programs where mature spermatozoa could not be isolated from testicular biopsies. The details of the indications for spermatid insemination, the technicity of the procedure and the results are exposed.

[Andrologic/gynecologic microsurgery and fertilization in vitro: complementary approaches?]. / Microchirurgie andrologique/gynécologique et fécondation in vitro: approches complémentaires?

The authors give their detailed results of andrological and gynecological microsurgical procedures and compare these to the cumulative results of their IVF work. They do defend the idea that to abandon microsurgery in favour of IVF and its last developments such as MESA & TESE is unreasonable and believe that every case demands a precise evaluation in which the gynecological situation and the age of the partner is mandatory.

Fertilizing ability of immotile spermatozoa after intracytoplasmic sperm injection.

Sometimes spermatozoa from ejaculate, epididymis or testis show a total absence of motility. For some patients, however, very few spermatozoa with very poor motility can be found after several hours of incubation (initially immotile spermatozoa). Other samples show no motility at all even after extended culture (totally immotile spermatozoa). Intracytoplasmic sperm injection (ICSI) is the only method available to select and retrieve a single immotile or initially immotile spermatozoon and inject it into the oocyte. A total of 103 patients with asthenozoospermia underwent ICSI in this study. It was shown that initially immotile and totally immotile spermatozoa, whatever their origin, have the capacity to fertilize an oocyte after ICSI. No significant difference could be observed between the fertilizing capacity of testicular or epididymal spermatozoa. Totally immotile ejaculated spermatozoa, however, fertilized significantly fewer oocytes after ICSI when compared with initially immotile ejaculated spermatozoa. Embryos of lower quality tended to be produced when totally immotile spermatozoa of any origin were used, compared with embryos resulting from initially immotile spermatozoa. Ongoing pregnancies were conceived after ICSI with initially immotile spermatozoa from any origin and totally immotile spermatozoa retrieved from testis only. One biochemical pregnancy was the result of embryo transfer after ICSI with totally immotile ejaculated spermatozoa. No supernumerary embryos could be cryopreserved for patients with totally immotile spermatozoa from ejaculate or epididymis. For a Kartagener patient, subzonal insemination (SUZI) seemed to be a better approach for obtaining fertilization and pregnancy than ICSI because no fertilization occurred after ICSI on sibling oocytes. Hence a healthy pregnancy was obtained after SUZI.

New therapeutic possibilities handling residual azoospermia.

Obviously, medical therapy of secretory azoospermia or microsurgical therapy of excretory azoospermia are not always successful. The unsolvable cases therefore can be grouped as residual azoospermias. Both the medical and microsurgical approaches are reviewed and their success rates analyzed. Some problems are unsolvable after work-up of the diagnosis. Till the early nineties, such patients were discouraged to undergo further medical therapeutic approaches and were advised to consider either adoption or donor insemination. At the present time, new possibilities have risen since the use of epididymal spermatozoa for performing assisted fertilization has considerably altered the picture. Furthermore, the last breakthrough of using testicular spermatozoa combined with the ICSI procedures have offered solutions that were unthinkable only a few years ago. The impact of these new approaches is discussed and the future development of microsurgery versus assisted reproduction techniques is also considered.

Pregnancy obtained with human testicular spermatozoa in an in vitro fertilization program.

The fertilizing capacity of human testicular spermatozoa and the positive outcome of an in vitro fertilization program open a wide range of opportunities for men suffering from obstructive and inoperable azoospermia. In six cases, subzonal sperm injection or intracytoplasmic sperm injection techniques were applied to inject testicular spermatozoa into human oocytes. The fertilization rate after testicular sperm injection reached 45%. Normal cleavage was observed and replacement of 10 embryos in 6 patients resulted in one chemical and one ongoing pregnancy.

Prevention of multiple pregnancies in an in vitro fertilization program.

OBJECTIVE: To limit the high number of multiple pregnancies in an IVF program. SETTING: In Vitro Fertilization Laboratory, Fertility Department, Public Hospital. INTERVENTIONS: The number of embryos transferred was limited to two instead of three. RESULTS: Limiting the number of embryos transferred to only two did not influence the take home baby rate but eliminated triplet and quadruplet gestations. Moreover, the number of patients with good quality supernumerary embryos available for cryopreservation increased. CONCLUSIONS: To reduce the high frequency of multiple gestations in an IVF program, the number of embryos replaced should be limited to a maximum of two.

A monozygotic twin pregnancy after application of zona rubbing on a frozen-thawed blastocyst.

Assisted hatching techniques enhance the success rate of implantation of in-vitro fertilized human embryos. We report here the successful transfer of a frozen-thawed human blastocyst on which we applied a non-invasive zona rubbing technique (reduction of the thickness of the zona pellucida by gentle rubbing with a microneedle). The implantation in the uterus led to the delivery of healthy monozygotic twins.

From oral contraception to hormone replacement therapy: towards a continuum?

PIP: On the basis of available evidence, it is reasonable to conclude that at this time women between 35-45 years should not be denied the benefit of oral contraception (OC) if they do not smoke. As Upton recently reported, the risk of death due to pregnancy and childbirth, even in a developed country such as the US, is greater than the risk of OC, including the risk for OC users who smoke. Low-dose, or very low-dose, ethinyl-estradiol combined OCs most likely can be prescribed safely for most women up to the time of menopause in the absence of cardiovascular risk factors. The alternative treatments that might be initiated before and then continued during and after the climacteric include: cyclic or continuous combined estrogen-progestogen preparations containing estradiol (in valerate or micronized form); "transdermal therapeutic systems" delivering both estrogen and progestogen, for cyclic or even continuous use; and other newly-developed means of delivering fairly constant doses of steroids, such as pellet implants and microspheres. The combination of estradiol pellet implants with the cyclic or continuous administration of progesterone or a progestogen also might prove to be a promising approach if estrogen accumulation could be avoided. Substantial effort still needs to be made to improve the available preparations and provide the clinician and the women concerned with the best possible formulations for use in the perimenopause, and possibly indefinitely afterwards as true substitution therapy.^ieng

Within-subject variations of sperm parameters and their importance in an in vitro fertilization program.

Spermiograms of 150 males were assessed, in view of studying them before an in vitro fertilization attempt. Within subject variations were determined with regard to sperm count and spermatozoal motility pattern. Ultimately these results were compared to the seminological parameters studied at the time of the in vitro-fertilization attempt. Overall results showed a clear drop in semen parameters at the time of egg retrieval. This drop was more marked when semen parameters were amongst the higher values at the start.