Anne-Marie Junca

Cryopreservation of immature and mature hamster and human oocytes.

The introduction of superovulation techniques into human in vitro fertilization (IVF) raised the problem of the production of large numbers of oocytes and embryos. The development of embryo cryopreservation offered an alternative to egg discarding and became successful in many IVF groups.- In the same way freezing also represented a way of storing excess human oocytes. Oocyte cryopreservation, first successful in mouse,6 hamster, rabbit, and monkeyI6 was applied in human^^^-^ and led to the first birth after in vitro fertilization of frozen-thawed (IT) oocytes. However, the risk of increased aneuploidy resulting from exposure of mature eggs to freezing and thawing was of concern. The cold treatment is known to cause depolymerization of microtubules and disappearance of microtubule-organizing centers in rabbit: mouse,2 and sheep22 oocytes. At this stage, chromosomes of the second meiotic division are attached to the cold-sensitive spindle microtubules. Any disruption in this spindle may lead to oocyte chromatid nondisjunction at fertilization resulting in aneuploid embryos. In fact, a benefit effect of cryoprotective agents such as dimethyl sulfoxide (DMSO) on microtubule polymerization was reported: and GlenisterZ3 did not find any increased incidence of aneuploidy in mouse embryos derived from frozen-thawed eggs. Nevertheless, it seemed attractive to freeze immature dictyate oocytes, thus avoiding

Transvaginal sonographically controlled ovarian puncture for oocyte retrieval for in vitro fertilization

Two hundred twenty-two patients took part in a trial of follicle puncture via the transvaginal route under sonographic control for the purpose of in vitro fertilization (IVF). Induction protocols were mainly human menopausal gonadotropin (hMG)+human chorionic gonadotropin (hCG) and clomiphene + hMC + hCG. In 79.7% oocyte aspiration could be achieved without difficulty via the transvaginal route. An average number of 4.7 oocytes per attempt was obtained: 10.7% evolutive pregnancies were obtained. No major incident was noted. This technique offers several crucial advantages: it reduces surgical risk, reduces the length of the patients stay in hospital as well as the overall cost of the procedure, and it also makes possible puncture in some cases hitherto regarded as excluded.

Morphologic and Cytologic Study of Human Embryos Obtained by In Vitro Fertilization

Among the main reasons for implantation failure of human embryos obtained by IVF, the embryo viability is the most difficult parameter to analyse. Indeed, as embryos are replaced at the two- or four-cell stage, nothing is known about their developmental capacity. For this purpose, we investigated the morphologic and cytologic aspects of the embryos obtained during the last year.

Impairment of Human Embryo Development after Abnormal in Vitro Fertilization

Normal in vitro fertilization (IVF) of human oocytes is now well established: an optimal fertilization and cleavage rate of 80% is reported by many teams, and about 400 children have been born thus far by means of this technique. However, careful examination of oocytes a t the pronuclear stage can show some abnormalities in the fertilization process. In fact, the relation between the delay in fertilization and an increase in abnormal development has been convincingly demonstrated in mammals and is often expressed by chromosomal abnormalities such as triploidy, monosomy, and trisomy. In vitro aging of rabbit oocytes 12 hours prior to fertilization was associated with a variety of anomalous developmental patterns including triploidy (6% of the eggs) and polyspermy (8% of the specimens). Approximately 10% of the eggs were artificially activated rather than fertilized, that is, they contained subnuclei and displayed no evidence of sperm penetration. Increasing the delay of fertilization increases the rate of activated oocytes up to 75% after a 24-hour aging period. As a consequence of these findings, we examined the incidence of fertilization abnormalities in our IVF program and their effects on early embryonic development.

The Implantation Window in Humans after Fresh or Frozen-Thawed Embryo Transfers

In mammals, the opdmum time for embryo transfer greatly depends upon the species. In rodents, a high synchrony between the donor and the recipient is required.1,2 In sheep3 and catde4 too, better results are obtained with synchronous transfers. In other species, such as pigs5, mares6 and monkeys7, an asynchrony up to one or even two days is possible.

Receptive and Refractory Period in Human Implantation

The successful establishment of pregnancy after embryo transfer requires a healthy blastocyst and a uterus that accepts it.

A function test to assess the responsibility of oocyte and sperm quality in in vitro fertilization failure.

It is obvious that both oocyte and sperm quality play a major role in the success of in vitro fertilization (IVF). In the case of total fertilization failure (TFF), and mainly for idiopathic infertility, it is often difficult to attribute the failure to either sperm or oocyte accurately. Indeed, oocyte quality is a parameter very difficult to appraise. A few criteria can easily be taken into account noninvasively, i.e., cumulus cell dispersal, nuclear maturity assessed by the presence of the first polar body at the time of insemination, absence of any sign of atresia, and sperm-zona pellucida binding, the day after insemination (1). When observing oocytes by transmission electron microscopy, abnormalities were sometimes evident in the cytoplasm of mature oocytes, such as delayed formation of aggregates of the smooth endoplasmic reticulum, demonstrating some degree of asynchrony between nuclear and cytoplasmic maturation and showing that nuclear maturation is not a sufficient criterion to appraise the capacity of oocytes to be fertilized (2). Certain characteristics of the follicular fluid biochemistry such as al-antitrypsin, proteoglycan, and immunoglobulin levels can also reflect differences in oocyte maturity and fertilizability (3). Moreover, a comparison of the basal secretion of steroids (progesterone, testosterone, and estradiol) in cumulus cells of different types of cumulus--oocyte complexes confirmed that oocytes recovered after a stimulation treatment consist of a heterogeneous population, and in fact they differ in their potential for fertilization and consequent cleavage (4). If we are at a loss when facing the problem of oocyte quality, the situation is even worse and more complex in the case of sperm quality. There is now a consensus on the fact that spermiocytograms do not predict sperm potential. Indeed, we occasionally obtained fertilizations with no more than 500,000 motile sperm/ml in the ejaculate. A battery of tests, such as the sperm penetration assay (SPA) (5), the hypoosmotic sperm swelling test (6), and the hemizona assay (7), have all shown various degree of correlation with oocyte fertilizability. The most widely applied test in clinical practice, i.e., the sperm penetration assay, has an advantage compared to biochemical assays: it gives information about physiologically functional acrosome reactions and sperm fusiogenic properties, prerequisite for fertilization. It is, however, time-consuming and costly, requiring the maintenance of an animal breeding unit and a special laboratory with trained staff. Sperm penetrat ion assays using human oocytes that failed to fertilize in vitro were developed recently as an alternative to SPA. Either saltstored (8), intact (9,10), zona-free (9-11), or cryopreserved (12), these oocytes seem to provide an accurate system to assess sperm function and fertilizing potential. We report here on a test developed to appraise the respective roles of oocyte and sperm quality after fertilization failure in couples with either idiopathic or male infertility, by reinseminating unfertilized eggs with sperm from a fertile donor.

From Oocyte to Embryo: A Model, Deduced from in Vitro Fertilization, for Natural Selection against Chromosome Abnormalities

A cytogenetical analysis was performed on 151 unfertilized oocytes, 22 fertilized eggs at the pronuclear stage, and 108 cleaved embryos obtained in the course of in vitro fertilization (IVF). Thirty-two per cent of unfertilized oocytes were abnormal, carrying nullisomies or disomies, mainly of D and G chromosomes, and a structural anomaly (Gq-) in one case. Fertilized eggs showed frequent asynchronism in the development of pronuclei and only 2 out of 8 karyotyped pronuclei were normal. Cleaved embryos were classified according to the number of pronuclei observed 17 hours after insemination. One per cent displayed a single pronucleus, and haploid chromosome complements were found in the corresponding cleaved embryos which were considered to be parthenotes. The rate of chromosome abnormalities of diploid eggs depended on their morphological aspect. Healthy cleaved embryos carried 12.5% of anomalies while this rate reached 37% in fragmented embryos (p less than 0.05). Lastly, 6% of fertilized eggs displayed three pronuclei or more. Only 41% of the corresponding embryos were triploid. Diploidy or diploidtriploid mosaicism were often encountered. This leads to a 21% rate of abnormalities in the preimplantation embryos. Parental karyotyping and HLA typing were carried out in a series of eight couples with in vitro idiopathic infertility or recurrent embryo degeneration in vitro. No abnormality was noted. According to these results, a model of natural selection of normal conceptuses is proposed.

Sequential use of clomiphene citrate, human menopausal gonadotropin, and human chorionic gonadotropin in human in vitro fertilization. I. Follicular growth and oocyte suitability**Supported by the Institut National de la Santé et de la Recherche Médicale (INSERM).

Sequential treatment with clomiphene citrate, human menopausal gonadotropin, and human chorionic gonadotropin was successfully applied to 149 women in the framework of an in vitro fertilization and embryo transfer program and led to the birth of normal children. An average of 3.5 follicles was promoted at each cycle. Oocyte maturity was evaluated at recovery either histologically or with respect to cumulus-oocyte complexes (COC) and correlated to oocyte fertilizability. Three types of COC are described according to cell dissociation and mucification. The most expanded cumulus mass (type I) was highly fertilizable, compared with granular and poorly dissociated cumulus complexes (types II and III, respectively). No improvement in the type II and III oocyte fertilizability was obtained when type I follicular fluid was added to the incubation medium. Moreover, incubation of type III oocytes in medium containing type III follicular fluid seemed to decrease their fertilizability.

Acrosome reaction of human sperm used for in vitro fertilization***Supported by INSERM PRC 135047.

The acrosomal reaction of human spermatozoa was assessed, according to a triple-stain technique, at the end of a 17-hour capacitation period in the search for a predictive test of sperm efficiency in an in vitro fertilization program. A 400% increase in the acrosomal reaction rate of the living spermatozoa was observed (4.2% +/- 3.2% before incubation versus 16% +/- 11% after incubation). No correlation could be evidenced between the abilities for spermatozoa to react and to fertilize mature human oocytes in vitro, whatever the initial semen quality. The acrosome reaction rate was not enhanced by the presence of the cumulus oocyte complex, by the selection of motile spermatozoa, or by the increase in albumin concentration of the culture medium.