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.

In vitro fertilization, development, and implantation after exposure of mature mouse oocytes to visible light.

Mature mouse oocytes were exposed prior to in vitro fertilization to visible light during 1, 2, or 4 hr at an intensity of 4,000 lux. Compared to controls cultured under identical conditions but protected from light, exposed eggs did not show any significant modification of cleavage speed and rate. After transfer of blastocysts obtained in vitro in uteri of pseudopregnant females, the implantation rate and the proportion of normal fetuses were not found to be different in relation to preliminary light exposure of oocytes fertilized and cultured in vitro.

Towards the definition of the fertilizing spermatozoa.

The seminological work-up of sperm samples is today totally different than only 20 years ago and the new more detailed readings bring the seminologist closer to the definition of the fertilizing capacity of sperm samples. Still, however careful the descriptive semen analysis, in 10% the predictive value is wrong. In view of inadequacies of the descriptive semen analysis, attempts to improve the laboratory contributions have focussed on the development of more refined techniques concerning the functional competence of human spermatozoa: computerized study of sperm motility, zona-free hamster oocyte penetration assay, hemizona-binding assay, study of the acrosoma reaction, biochemical studies. But it must be considered that the cost of all sperm controls in order to evaluate its fertilizing capacity can become on even harder financial burden than on IVF attempt itself.

[Assistance in human oocyte fertilization using micromanipulation]. / L'assistance à la fécondation de l'ovocyte humain par micromanipulation.

In recent years different types of micromanipulation have become available to improve human gametes interaction, particularly in cases of severe sperm defect. Direct injection of one spermatozoon into the cytoplasm of the egg has not been applied in man, not only because of technical difficulties and low efficacy, but also in view of the theoretical risk of chromosomal anomalies. However, subzonal sperm injection (SUZI) and partial zona dissection (PZD) are now used routinely by a number of IVF centers. In our hands this latter method has increased significantly the fertilization rate (21.6 vs 2.9 p.cent among intact oocytes) and has allowed to obtain 3 pregnancies (of which 2 ongoing) after 28 attempts. Our results and those of the literature are discussed with respect to the risks and advantages linked to these techniques.

Improvement of fertilizing performance by normal and abnormal mouse semen after zona opening of mature oocytes.

The usefulness of opening the zona pellucida by partial zona dissection (PZD) to enhance fertilization of mature mouse oocytes was studied after insemination with three types of semen: normal and diluted semen and semen from long-term-vasectomized males. Zona opening did not by itself induce parthenogenetic cleavage of mature oocytes and did not significantly increase mono- and polyspermic fertilization of oocytes inseminated with normal semen. While a fertilization rate of 62% was obtained among intact oocytes, of which 4.5% were polyspermic, a 66.8% fertilization rate was observed among PZD oocytes, 6.3% of which were polyspermic. However, after using diluted semen, only 54 of 193 intact oocytes were fertilized (28%), and PZD improved the fertilization rate to 65.4%. Cleavage rate of nonmanipulated oocytes inseminated with abnormal semen from vasectomized males was dramatically decreased in comparison with those inseminated with normal semen (7.6% vs. 65%). PZD induced a moderate but significant improvement of fertilization performance when using this abnormal semen (19.6%).

Sperm morphology and IVF pregnancy rate: comparison between Percoll gradient centrifugation and swim-up procedures.

Many groups currently use two methods for the separation of motile spermatozoa, swim-up (S-up) and centrifugation on discontinuous Percoll gradient (PGC), and comparison of results indicates that PGC is superior. In this study we have attempted to identify the factors explaining this difference. This laboratory has long-standing expertise in seminology, thus the parameters of sperm morphology were the obvious first choice for detailed study. First, the respective effects of S-up and PGC on sperm morphology were analysed in different types of ejaculates: 62 semen samples with normal parameters and 41 with poor parameters. Both separation techniques resulted in improved morphology in the final preparation but only the increase of morphologically normal spermatozoa in the final Percoll suspension was significant. Second, application of these techniques in our in-vitro fertilization (IVF) programme revealed that, together with the improvement of sperm morphology, a higher pregnancy rate was obtained after PGC. The ongoing pregnancy rates per oocyte retrieval were 21.1% for the S-up technique and 33.3% for the PGC technique. These data show that spermatozoa selected by PGC present an improved morphology which we believe to be linked to improvement of the quality of the in-vitro fertilized embryos and ultimately the percentage of successful IVF results.

Techniques of sperm selection, improvement and separation in an in vitro fertilization program.

The Authors present a review of the various techniques used for improving the fertility potential of sperm within the context of an in vitro fertilization (IVF) program. After a brief description of the mechanisms leading to normal in vivo fertilization, they discuss the different methods of selecting and improving sperm for IVF. They conclude that centrifugation on discontinuous Percoll gradients would seem to be the most efficient separation method from all points of view, while the addition of pharmacological agents to improve sperm quality and motility lead to extremely unsatisfactory results.

Some factors affecting successful vitrification of mouse blastocysts.

The effects of temperature and exposure time to vitrification solutions on In vitro survival of mouse blastocysts were investigated. Blastocysts were first exposed for 10 min to vitrification Solution 1 (VS1) containing 10% glycerol-20% 1,2 propanediol in phosphate buffered saline (PBS), then to vitrification Solution 2 (VS2) with 25 % glycerol-25% 1,2 propanediol for various periods either at room temperature or at 4 degrees C. At room temperature survival dropped quickly, while at 4 degrees C an increase in survival was observed. It is concluded that the viability of mouse blastocyts after vitrification is dependent on the temperature and duration of equilibration in vitrification solutions.

Determination of porcine plasma follitropin levels during superovulation treatment in cows.

Porcine follicle stimulating hormone (pFSH) and porcine luteinizing hormone (pLH), are widely used to induce superovulation in cows. An advantage of this treatment is that the LH:FSH ratio can be varied to optimize the growth of the ovarian follicles. However, due to the relatively short half-life of FSH, the superovulatory treatment requires numerous injections. A performant radioimmunoassay system (sensitivity=0.2 ng/ml plasma) was used to determine plasma pFSH levels in cows that were superovulated with 2 daily injections of 4 Armour Units (A.U.) of pFSH for 4 d. From plasma profiles, the half-life and the disappearance of pFSH were estimated at 5 h and at 10 to 12 h, respectively, confirming the necessity of using two daily injections.

Effect of stage of development on survival of mouse embryos frozen--thawed rapidly.

Embryos were recovered on Day 4 of pregnancy from superovulated random-bred OF1 Swiss albino mice. They were classified into four categories based on their stage of development: expanding blastocyst, blastocyst, early blastocyst, and compacted morula. They were then cooled at 2 degrees C/min from -7 to -25 degrees C in a freezing medium containing 1.36 M glycerol and 0.25 M sucrose in phosphate-buffered saline (PBS). At -25 degrees C, they were plunged into LN2 and thawed a few hours later in water at 20 degrees C. After washing in PBS, recovered embryos were cultured for 20 to 24 hr and the number of embryos that had developed normally was recorded. The results showed a clear effect of the stage of development on survival. Survival of expanding blastocysts and blastocysts was very low (1.4 and 21.8%, respectively) compared to that of early blastocysts and compacted morulae (69.4 and 73.5%). The more differentiated stage of the blastocyst (two kinds of cells) and the presence of a blastocoelic cavity may explain the differences observed under our cooling conditions. As a further test of viability, 93 blastocysts that had developed in culture for 20 hr from 153 frozen-thawed early blastocysts and compacted morulae (60.8%) were transferred to 8 recipient mice. Seven became pregnant, yielding 38/82 normal live young (46.3%).

Effects of in-vitro fertilization, culture, freezing and transfer on the ability of mouse embryos to implant and survive.

The survival after transfer of frozen-thawed mouse blastocysts obtained from culture of in-vitro fertilized oocytes or 1- and 2-cell ova was compared. About 10% of transferred embryos developed to term in each group and there was no difference between embryos fertilized in vitro or in vivo. In addition to embryonic loss due to transfer, in-vitro cultivation and freezing reduced the proportion of fetuses considered viable at Day 15 of pregnancy (29.8 versus 50.7% and 26.3 versus 50.7% respectively). When used together these procedures had an additive effect on fetal wastage (18.4 versus 50.7%). In-vitro culture also entailed a significant increase of resorbing implantation sites (10.2 versus 4.3%). The re-expansion rate after freezing and thawing of blastocysts grown in vitro was paradoxically greater than that of blastocysts grown in vivo (85.8 versus 54.6%).

Fast freezing of cow embryos in French straws with an automatic program.

Cow embryos between day 6.5 and 9 were frozen in 1.5M DMSO in PBS at 2 degrees C/min from seeding to -25 degrees C before being plunged into liquid nitrogen directly or after 10 min at -25 degrees C. Cooling rate from 20 degrees C to -5 degrees C was 9 degrees C/min. Seeding was induced automatically at -5 degrees C by injection of liquid nitrogen vapour. Embryos were subsequently thawed by direct transfer to water at 20 degrees C (group I) or at 37 degrees C (group II). Survival was assessed by culture in vitro and by transfer. In group I, 35.7% were degenerated after thawing (compared to 35.4% in group II). Survival rate after culture in vitro for 24h was not significantly different (48.3% vs 42.8%) and hatching rate after 96h culture was quite similar (33.3% vs 34.4%). In group II, four pregnancies were obtained from 10 embryos transferred. Time at -25 degrees C did not improve the results. Automatic seeding did not impair survival. These results show that the quality of the embryo is the determinant factor for survival after freezing and that the plastic straw is the most suitable vessel for freezing, storage and transfer of embryos.

Deep freezing of cattle embryos in glass ampules or French straws.

Ninety four cow embryos recovered on day 7-8 after onset of oestrus were frozen by the "Two Step" freezing procedure: 49 in pyrex glass ampules and 45 in .25 ml French semen straws. The overall survival rate was 33.7% (36.2% for embryos frozen in glass ampules; 31.1% for embryos frozen in plastic straws). 45.2% of transferred embryos resulted in pregnancies (35.7% after freezing in glass ampules v.s 52.9% after freezing in plastic straws).