GnRH agonist versus GnRH antagonist in oocyte donation cycles: a prospective randomized study.

BACKGROUND: The specific role of LH in folliculogenesis and oocyte maturation is unclear. GnRH antagonists, when administered in the late follicular phase, induce a sharp decrease in serum LH which may be detrimental for IVF outcome. This study was performed to evaluate whether the replacement of GnRH agonist (triptorelin) by a GnRH antagonist (ganirelix; NV Organon) in oocyte donation cycles has any impact on pregnancy and implantation rates. METHODS: A total of 148 donor IVF cycles was randomly assigned to use either a GnRH antagonist daily administered from the 8th day of stimulation (group I) or a GnRH agonist long protocol (group II) for the ovarian stimulation of their donors. The primary endpoints were the pregnancy and the implantation rates. RESULTS: The clinical pregnancy rate per transfer (39.72%, 29/73 versus 41.33%, 31/75) based on transvaginal scan findings at 7 weeks of gestation, the implantation rate (23.9 versus 25.4%) and the first trimester abortion rate (10.34 versus 12.90%) were similar in the two groups. CONCLUSION: In oocyte donation cycles the replacement of GnRH agonist by a GnRH antagonist appears to have no impact on the pregnancy and implantation rates when its administration starts on day 8 of stimulation.

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.

Congenital malformations after intracytoplasmic injection of spermatids.

Spermatid microinjection into oocytes was applied in cases of intracytoplasmic sperm injection (ICSI)/testicular sperm extraction (TESE) where no spermatozoa could be found in numerous testicular samples. Although several pregnancies were obtained with this procedure, serious concerns remain regarding its safety. Although the relevance of the injection of spermatids is by no means certain, we wish to report that from four pregnancies obtained after injection of elongated spermatids, two cases of major malformation resulted.

Oocyte insemination with spermatozoa precursors.

Since the use of testicular spermatozoa in programs of assisted fertilization proved very successful, attention was focussed on the use of spermatids also carrying 23 chromosomes. Several difficulties became obvious; the first one concerned the recognition of round spermatids. This is a problem which does not concern elongating and elongated cells. The intra-cytoplasmic injection of elongated spermatids resulted in several pregnancies but this is not so for the round ones. Although, in the group of patients in whom only round spermatids are found at the time of the attempt, is to be divided into two categories; patients in whom previous research allowed to find spermatozoa, however few, and patients who never produced spermatozoa at all. This last group is no longer an indication for intracytoplasmic sperm injection procedure unless in the future new culture media allow a maturation into elongated forms.

Successful treatment of idiopathic anejaculation with electroejaculation after microsurgical vas aspiration.

This case report describes a couple suffering from infertility secondary to psychogenic anejaculation, which was refractory to all conservative treatment modalities. A first trial of microsurgical vas aspiration in combination with in-vitro fertilization (IVF) resulted in a pregnancy. After 2 years, three more trials of microsurgical vas aspiration in combination with either IVF or subzonal insemination (SUZI) resulted in embryo transfer without pregnancy. Finally, after 3 years, spermatozoa obtained by rectal probe stimulation under general anaesthesia were cryopreserved. A second intracytoplasmic sperm injection (ICSI) procedure using these cryopreserved spermatozoa also resulted in a second pregnancy. Although sperm concentration was in the normal range, in all samples obtained by either rectal probe electrostimulation or microsurgical vas aspiration, motility was <30% in all but two samples.

Is there a future for spermatid injections?

Microinjection of spermatids into oocytes has proven to be a successful assisted reproduction procedure in the animal model. In the human, low fertilization and cleavage to the 4-cell stage were reported after intracytoplasmic sperm injection (ICSI) with round spermatids. In comparison with a conventional ICSI-testicular sperm extraction (TESE) programme, the implantation rate after round spermatid injection is dramatically low. Different problems have been encountered during the development of the spermatid injection technique and they could be partially responsible for the lower outcome when using round spermatids. Compared with the round spermatid cells, spermatids in the elongation phase are easy to isolate and identify from other round cells present in a wet preparation. The morphological identification does not reveal anything about the viability or the genetic normality of the round spermatids. Severe testicular dysfunction may have consequences on the quality of the few spermatogenic cells present. Others factors, such as the pathology of the patient, play an important role in the successful treatment. Even if the results are extremely low, spermatid injection seems more favourable for men who have already proven their capacity to produce some spermatozoa. A spermatogenic block at the round spermatid level has led to early abortions, increasing the suspicion of the role of a genetic factor. In order for this technique to be safe for use in clinics, more intensive work is needed to improve the selection and handling of cells and to ascertain the genomic imprinting and gene expression necessary for embryonic development. Hence, when using immature cells for conception, the screening of the patient and the follow-up of the pregnancies and babies should be mandatory.

Relationship of human follicular diameter with oocyte fertilization and development after in-vitro fertilization or intracytoplasmic sperm injection.

The aim of this work was to evaluate the relationship between follicular size at the time of oocyte retrieval, and the subsequent oocyte competence to be fertilized and to develop in vitro. All the obtained oocytes were classified according to the corresponding volume of aspirated follicular fluid. Aspirated volume of follicular fluid <2 ml corresponded to a follicular diameter <16 mm and constituted the small size group. Volume of follicular fluid from 2 to 6 ml corresponded to a diameter from 16 to 23 mm and constituted the medium size group. The large size group contained follicles with diameter >23 mm and corresponded to an aspirated volume of follicular fluid of >6 ml. A progressive and significant increase in the rates of oocytes with a first polar body was observed from the small size group to the other groups and from the medium to the large size group: 75.3, 85.9 and 95.3% respectively. After classical in-vitro fertilization (IVF), significantly better rates of fertilization and development were obtained in the medium size group compared to the two other groups. Moreover, a positive relationship was observed between follicular diameter and rates of embryos scored as 'good' when oocytes were fertilized by intracytoplasmic sperm injection (ICSI). These results demonstrated that follicular size is positively related to the oocyte ability to be fertilized and to develop. Although oocytes from small follicles gave lower percentages of development probably due to partial oocyte incompetence, they allowed an increase in the total number of embryos scored as 'good'.

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.

Pregnancies achieved with testicular and ejaculated spermatozoa in combination with intracytoplasmic sperm injection in men with totally or initially immotile spermatozoa in the ejaculate.

The efficacy of intracytoplasmic sperm injection (ICSI) employing testicular and ejaculated spermatozoa was assessed in 24 couples with totally or initially immotile spermatozoa. No criteria were employed in selecting which patients would be treated with testicular or ejaculated spermatozoa. The men were chosen at random. Testicular spermatozoa obtained by testicular sperm extraction were used in 14 and ejaculated spermatozoa were used in 10 of these couples. In all cases. asthenozoospermia was total in their basal semen sample. In 12 male partners, spermatozoa were totally immotile before and after Percoll gradient fractionation (totally immotile). In the remaining 12 men, spermatozoa initially showed a total absence of motility; however, some of the spermatozoa had showed very poor motility (0. 1%) after Percoll gradient fractionation and a 1.5-2.0 h incubation period (initially immotile). Of these 24 total asthenozoospermic males, 14 also had total teratozoospermia. The fertilization and cleavage rates in the testicular and ejaculated sperm groups were 53. 5 and 96.3 and 54.5 and 94.4% respectively. One cycle resulted in complete fertilization failure, and in 23 embryo transfer cycles a total of 10 pregnancies were obtained (41.6%). Eight pregnancies were achieved in the testicular sperm group, while only two pregnancies were obtained in the ejaculated sperm group. Four pregnancies, two from the ejaculated sperm group and two from the testicular sperm group, resulted in clinical abortions in the first trimester. Of the remaining six pregnancies, two have already resulted in healthy births and four pregnancies are now in the second or third trimester in the testicular sperm group. Using testicular spermatozoa in combination with ICSI can be an alternative mode of treatment in cases with totally or initially immotile spermatozoa in the ejaculate. Very low pregnancy rates have been obtained and no ongoing pregnancy has been achieved using ejaculated spermatozoa in these cases.