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SummaryThe aim of this study was to evaluate the advantages of the two-step embryo transfer (ET) strategy combining a day 2/3 ET with a day 5/6 blastocyst transfer. In an observational comparative study, 400 infertile women were enrolled from two assisted reproductive technology (ART) units according to inclusion criteria: age below 42 years and at least three embryos obtained on day 2 thus allowing an extended in vitro culture. Two groups were defined according to the ET strategy adopted: group 1 had a two-step ET; and group 2 had a day 2/3 ET with (subgroup 2a) or without (subgroup 2b) blastocysts cryopreserved on day 5/6. Live birth rate was significantly higher in group 1 than in subgroups 2a and 2b (36.5% versus 29.4% and 13.4%, respectively; p < 10−3). Multiple pregnancy rates were comparable between groups. After adjusting on major prognostic factors, the two-step ET strategy was still associated with a significantly higher live birth rate than the day 2/3 ET (OR = 2.23; 95% CI: 1.32–3.77). The two-step ET provides better live birth rates than the cleavage-stage ET. It does not increase multiple pregnancy rates if the number of embryos transferred is limited. It also prevents cycle loss when embryos fail to develop into blastocysts.

Résumé Introduction L’analyse fine de la morphologie des spermatozoïdes à un grossissement de 6600 x, appelée MSOME (motile sperm organellar morphology examination) et appliquée en ICSI, a donné naissance à l’IMSI (intracytoplasmic morphologically selected sperm injection). Cette technique est proposée aux couples en échecs répétés d’implantation embryonnaire en ICSI, dans le but d’obtenir une grossesse évolutive. Matériel et méthodes L’étude concerne une cohorte observationnelle de 11535 ICSI pratiquées avec du sperme éjaculé frais, de janvier 2004 à juillet 2009. Parmi celles-ci, 2509 ont été réalisées avec IMSI. Les paramètres étudiés sont: le taux de clivage à J2 par ovocyte injecté, le taux de grossesses cliniques par ponction et le taux de fausses couches spontanées, en fonction du rang de la tentative et/ou de la qualité du sperme du bilan. Ces données ont été comparées entre l’ICSI et l’IMSI et les comparaisons ont été faites à l’aide de Chi2 et d’analyse de variance. Résultats Nous n’avons pas observé de différence significative entre l’ICSI et l’IMSI en termes de taux de clivage à J2 et taux de grossesses cliniques. Par contre, le taux de grossesses, en IMSI de rang 1, est significativement plus élevé en cas d’anomalie du sperme si on regroupe les tératozoospermies avec les oligozoospermies et les oligotératozoospermies (34,4 vs 27,1%, p = 0,02). De plus, si l’on regroupe les tératozoospermies et les oligotératozoospermies, le taux de fausses couches est plus faible en IMSI comparé à l’ICSI, de manière proche de la significativité (12,6% vs. 19,6%, p = 0,08). Conclusion En présence d’une tératozoospermie sévère, l’IMSI semble augmenter les taux de grossesses et diminuer les taux de fausses couches.

Resume L’analyse morphologique du spermatozoide (ou spermocytogramme) fait partie du bilan d’infertilite du couple et permet de definir la normalite d’un sperme ; cependant, la relation entre morphologie spermatique et fertilite est loin d’etre etablie. Bien que plusieurs etudes ne montrent pas de relation entre formes anormales et resultats de l’ICSI, il semble neanmoins que les taux de succes dependent de l’aspect du spermatozoide injecte. L’examen de la morphologie fine du spermatozoide (et notamment de la tete), a tres fort grossissement (de × 6600 a × 12500) (MSOME) en temps reel, permet de selectionner les plus normaux et de les injecter (IMSI). Cette selection permet d’augmenter, dans certaines situations, les taux de grossesse evolutive et d’implantation. Des criteres ultramorphologiques ont ete etablis et il semble que le plus predictif de la qualite du spermatozoide soit la presence de vacuoles dans la tete du spermatozoide. De plus, ces vacuoles semblent reliees a des alterations de l’ADN (fragmentation et/ou decondensation) ayant pour consequence des defauts du developpement embryonnaire. Pour objectiver les observations, beaucoup d’auteurs ont essaye de mettre au point des classifications et d’etablir un spermocytogramme a fort grossissement qui pourrait etre utilisable en routine et pourquoi pas dans l’avenir, remplacer le spermocytogramme.

Embryos are conventionally selected for transfer based on the evaluation of the cleavage speed and extent of blastomere fragmentation. Here we examined whether the predictive value of these criteria, as indicators of the chance of embryo implantation, can be further potentiated by adding previously described criteria reflecting the regularity of pronuclear development. In a group of embryos selected for transfer in 380 fresh embryo transfer cycles according to the conventional criteria, the transfer of only those embryos that developed from zygotes judged normal at the pronuclear stage (pattern 0) gave significantly higher pregnancy (44.8%) and implantation (30.2%) rates compared with the pregnancy (22.1%; P < 0. 05) and implantation rates (11.2%; P < 0.001) for the transfers of only those embryos that developed from zygotes judged abnormal (non-pattern 0). The transfer of only one pattern 0 embryo was sufficient for the optimal chance of pregnancy (no differences in pregnancy rates after transfer of one, two or three pattern 0 embryos), whereas the transfer of two pattern 0 embryos mostly resulted in a twin pregnancy. The inclusion of the criteria based on pronuclear morphology can thus lead to the application of a single embryo transfer policy and optimize the selection of embryos for transfer and cryopreservation.

Human embryo cryopreservation represents an indispensable extension of in-vitro fertilization (IVF) programmes as long as they are based upon the recovery of a large number of oocytes. The most widely used procedures include the cryopreservation of human zygotes or embryos in early cleavage, using 1,2-propanediol and sucrose as cryoprotectants. Our results over a 10 year period (1986-1995) on 5032 thawed cycles involving 14 222 stored embryos make it possible to appraise the results and the contribution of embryo freezing to assisted reproduction. Embryos survived the freeze-thaw process in 73% of cases leading to 4590 transfers of 2.2 embryos (91% of thawed cycles). The clinical pregnancy rate per transfer was 16%, the live birth rate 12%, and the rate of babies born alive per transferred embryo was 6%. Embryo freezing monitored 10 years later produced an average of 8% of additional births. By then, 86% of stored embryos had been thawed for transfer to patients. Destruction or donation were required for only 8% of all frozen embryos and there was no news from the parental couple in relation to almost 6% of embryos. The fate of the vast majority of embryos was decided during the first 5 years of storage. Blastocyst cryopreservation is making new strides, thanks to co-culture systems and embryo selection. Micromanipulation procedures seem to have little impact on the outcome of embryo freezing. Human oocyte freezing is again clinically applied. Indeed, much of the concern about injuries to the oocyte structures through the freeze-thaw process do not seem to be justified, and the problems with frozen-thawed oocyte fertilization has been overcome using intracytoplasmic sperm injection (ICSI). As long as oocyte in-vitro maturation is not well controlled, better results will probably be obtained with mature oocyte cryopreservation. Emerging methods include the freezing of immature oocytes, follicles and ovarian tissue.

The expression of both components of the high-affinity leukaemia inhibitory factor receptor, LIFR beta and glycoprotein 130 (gp130), was investigated in human oocytes and individual in-vitro cultured preimplantation embryos by reverse transcription-polymerase chain reaction (RT-PCR). Messenger RNA of both LIFR beta and gp130 was detected in as little as 1/30 and 1/12 sample equivalents of cDNA respectively, in oocytes (n = 4), 4-cell and expanded, blastacyst stage embryos. LIFR beta but not gp130 transcripts were detected at the 2-, 8- and 10-cell stages, and in cavitating and hatched blastocysts. In order to exclude a simian origin of these PCR products resulting from the Vero cell line that was used as a feeder during culture to the blastocyst stage, they were digested with restriction endonucleases Taql (LIFR beta) or Kpnl (gp130). Their human origin was confirmed. The results support an earlier finding of LIFR beta mRNA expression in human blastocysts, and extend these results to earlier stages and oocytes. This is the first report of LIFR beta and gp130 transcription in human oocytes. Taken together these results demonstrate that transcription of LIFR beta and gp130 takes place throughout human preimplantation development, and suggest that functional LIF receptors might be present at these stages. These results further confirm the feasibility of performing mRNA phenotyping of multiple genes with RNA derived from a single preimplantation stage embryo.

La culture prolongee correspondait au depart a une necessite : les biotechnologies chez les animaux domestiques a interet zootechnique necessitent, pour le transfert embryonnaire, un transfert au stade blastocyste. En effet, le transfert aux stades precoces, apres fecondation in vitro et culture de courte duree, n’est tout simplement pas possible : les embryons sont expulses de l’uterus. Seul le transfert de morula tardives (5 jours) ou de blastocyste est operant. Les problemes de culture de l’embryon du stade 1 au blastocyste se heurtent, dans de nombreux cas, a un blocage au moment du cycle cellulaire correspondant a la transition du controle maternel au controle embryonnaire (MZT ou maternal to zygotic transition) : souris : 2–4 C, homme : 4–8 C, vache, lapin : 8–16 C. Ce cycle cellulaire dure en moyenne 24 heures pendant lesquelles demarrent les premieres transcriptions embryonnaires ; l’embryon prend son autonomie par rapport aux reserves d’ARN et de proteines stockees pendant la croissance de l’ovocyte, qui se sont epuisees pendant les premieres divisions.

Depuis quelques annees, plusieurs publications ont mis en exergue l’avantage de l’examen du sperme au fort grossissement (× 6 600), permettant l’evaluation de la morphologie du noyau spermatique et la densite de sa chromatine en temps reel avant micro-injection ovocytaire. Ce procede est communement appele motile sperm organelle morphology examination (MSOME). La technique d’ICSI est appelee intracytoplasmic morphologically selected sperm injection (IMSI). En reproduction, nous deplorons helas plus d’echecs que de succes. Les causes des echecs sont multiples, d’origine maternelle mais aussi paternelle. En effet, un sperme inadequat peut conduire au pire a un defaut de fecondation ou de developpement embryonnaire precoce et au mieux a une implantation embryonnaire suivie d’une fausse couche.

Ten couples included in our in-vitro fertilization programme, selected because of one of more previous total fertilization failures or a low fertilization rate (< 20%), were entered in an experimental protocol of egg insemination on autologous granulosa cells. Half the oocytes from each patient were randomly assigned to either a control or a co-culture group. We observed no difference in the fertilization rate between the control (16.2%) and co-culture groups (12.1%). Only two couples benefited from this technique since fertilization was obtained only in co-culture. The poor efficiency of this protocol led us to propose the use of sperm micro-injection if all classical attempts to improve fertilization were unsuccessful.

Cyclic voltammetry was used to investigate the mechanism of anodic oxidation of four aromatic amines, together with the effect of the addition of bases (lutidine, sym-collidine, quinuclidine and hydroxide). Without a methoxy substituent in the para position of the phenyl group, the coupling of two radical cations occurs on the addition of weak bases, but a deprotonation is involved in the presence of hydroxide ion. This deprotonation occurs in all cases when a methoxy substituent is present on the phenyl group.

In a preliminary, unpublished randomized study conducted in 2000 on 39 patients, including a placebo group, it was observed that the addition of growth hormone (GH) during ovarian stimulation in patients with poor-quality oocytes increased the pregnancy rate. However, the results were not statistically significant due to the small number of patients in each group. A protocol with 8 IU GH was tested in 291 patients with three or more previous failures of embryo transfer for no clearly identifiable reasons. The analysis was restricted to patients receiving either recombinant FSH or human menopausal gonadotrophin (HMG) (n = 245). They were compared retrospectively to all patients with three or more failures during the same period of time but stimulated only with recombinant FSH or HMG, without GH, in an observational study design. Co-stimulation with GH gave better results in terms of number of oocytes collected and embryos obtained. Pregnancy rate per retrieval was higher than in the control group (25.7% versus 18.2%, P < 0.01) and reached a level similar to the one observed in the study centre for the whole population. Ovarian stimulation associated with GH can be proposed for patients with a history of repeated assisted reproduction failures. An improvement of cytoplasmic competence is proposed as an explanation.

Until now, the morphological sperm analysis (spermocytogram) allows to define sperm normality, but the relationship between sperm morphology and fertility is not yet assessed. Although several studies do not report any relationship between abnormal sperm morphology and ICSI results, nevertheless, the success rate of ICSI sems to be dependent on injected sperm morphological aspect. Detailed morphological sperm examination (especially sperm head) at high magnification (from x 6600 to x 12500) (MSOME) in real time allows to select the best spermatozoa before oocyte injection (IMSI). In some cases, implantation and ongoing pregnancy rates were improved with this sperm selection method. Ultramorphologic criteria were established and the most predictive factor of sperm quality is the presence of vacuoles in the sperm head. Those vacuoles appear to be related to DNA damage (fragmentation and/or denaturation) and affect embryo development. To standardize those observations, several authors tried to establish sperm MSOME classifications in order to be used in routine and to replace the conventional spermocytogram in the next future.

ICSI failures (essentially fertilization and implantation failures) can be due to the quality of the oocytes, the spermatozoon injected and its manipulation, uterine factors and paternal factors, such as DNA fragmentation of the spermatozoa. Pre-selection of the spermatozoon is the crucial phase for ensuring successful ICSI. The characteristics of the sperm must be checked under the microscope at a magnification of x 6600 as certain abnormalities, in particular nuclear vacuoles, cannot be seen at x 300. In our centre, we conducted a study on 72 patients which showed that with more than 30% of fragmented DNA, neither implantation or birth was achieved using ICSI. Performing IMSI on the same patients allowed us to obtain implantation rates of 17.4% with 30 to 40 % of fragmented DNA and of 33.3% above 40%. Births were achieved in 17.4% and 28.6% of cases respectively. IMSI has the advantage of allowing extremely careful selection of the spermatozoon microinjected, so that it is of normal shape, with the least vacuoles possible and a low DNA fragmentation rate. It is, however, a complicated technique that cannot be routinely performed.

Previous studies have shown that repeated intracytoplasmic sperm injection (ICSI) failures can be caused by a paternal effect. Other studies have suggested that ICSI results are compromised if morphologically abnormal spermatozoa are injected into oocytes. This study was undertaken to evaluate the usefulness of a high-magnification optical system to select spermatozoa to be used for ICSI (high-magnification ICSI) in couples with repeated conventional ICSI failures. Couples with two or more previous conventional ICSI failures underwent an additional conventional ICSI attempt, followed by a high-magnification ICSI attempt. The outcomes of the two sequential attempts were compared. In 72 of these patients, sperm DNA integrity was assessed. In the whole group of 125 couples with repeated ICSI failures, high-magnification ICSI improved clinical outcomes (pregnancy, implantation, delivery and birth rates) without affecting biological outcomes (fertilization and cleavage rates, embryo morphology). The improvement of clinical ICSI outcomes was evident both in patients with an elevated degree of sperm DNA fragmentation and in those with normal sperm DNA status. It is concluded that high-magnification ICSI improves clinical outcomes in couples with previous repeated conventional ICSI failures.

The expression of both components of the high-affinity leukaemia inhibitory factor receptor, LIFR beta and glycoprotein 130 (gp130), was investigated in human oocytes and individual in-vitro cultured preimplantation embryos by reverse transcription-polymerase chain reaction (RT-PCR). Messenger RNA of both LIFR beta and gp130 was detected in as little as 1/30 and 1/12 sample equivalents of cDNA respectively, in oocytes (n = 4), 4-cell and expanded, blastacyst stage embryos. LIFR beta but not gp130 transcripts were detected at the 2-, 8- and 10-cell stages, and in cavitating and hatched blastocysts. In order to exclude a simian origin of these PCR products resulting from the Vero cell line that was used as a feeder during culture to the blastocyst stage, they were digested with restriction endonucleases Taql (LIFR beta) or Kpnl (gp130). Their human origin was confirmed. The results support an earlier finding of LIFR beta mRNA expression in human blastocysts, and extend these results to earlier stages and oocytes. This is the first report of LIFR beta and gp130 transcription in human oocytes. Taken together these results demonstrate that transcription of LIFR beta and gp130 takes place throughout human preimplantation development, and suggest that functional LIF receptors might be present at these stages. These results further confirm the feasibility of performing mRNA phenotyping of multiple genes with RNA derived from a single preimplantation stage embryo.

Between 1990 and 1994, a clinical retrospective study has been carried out at Tenon Hospital on 1200 patients: during this periods. 4845 embryos have been cryopreserved and 31% of the patients had this procedure for their supranumerary embryos. The rate of implantation by embryo was 8% per transfer, comparable to those fresh embryos. Less than 1% of the embryos were abandoned. The contribution of cryopreservation to the IVF program is substantial, increasing pregnancy rate by 10%. Moreover, the rate of multiples pregnancies is significantly lower when implanting frozen thawed embryos (9.6%) vs fresh embryos (p0.001). There was no difference between frozen-thawed and fresh embryos, in the implantation rate by embryo, the mean gestational age, and birth weight of singleton, twin and triplet births. From a biological point de vue, a series of 3693 embryos, carried on the same period, in our center has showed that for the success of this procedure, the quality of the embryos was more important, than the duration of the storage. The incidence of major and minor congenital malformations was not different in the two groups of babies (less than 3%). But a retrospective analysis carried out on 84 children, showed 4 major abnormalities, after a follow up of 1 to 9 years. However these anomalies do not seem to have some evident correlation with the cryopreservation procedure; a larger series and a prospective study are needed to get significant results concerning the health of the children born after the procedure of cryopreserved embryos.

Many studies in IVF practice, have tried to assess the maturity and quality of oocytes prior to insemination and relate it to IVF efficiency and to the pattern of ovarian stimulation. They were based on the indirect evaluation of the aspect of the cumulus-corona-cell complex (CCC) which was rapidly shown to be poorly correlated to the oocyte nuclear and cytoplasmic maturity in stimulated cycles. For sperm microinjection procedures, oocytes need to be peeled off from any follicular cell through hyaluronidase action in order to gain an easy access to the zona pellucida and the ooplasm. If therefore becomes possible to precisely known at recovery the nuclear status of the oocyte cohort as well as the rate of degenerative gametes. Immature oocytes can be further matured in vitro. Moreover, the ICSI procedure (intracytoplasmic sperm injection) allows a direct assessment of the cytoplasmic maturation, whatever the maturity of the ZP and its receptors and of the plasma membrane. On a preliminary evaluation of 70 ICSI cycles performed in our collaborative group from september to november 1994 and leading to a 24 % pregnancy rate per oocyte pick-up we focused on the true maturation of the oocyte cohort, its outcome and correlation with ICSI efficiency and stimulation protocol. On the 760 oocytes, 13.9% were atretic, 9% at the germinal vesicle stage (GV), 3.4% in metaphase 1 and 73.7% in metaphase 2 at recovery.(ABSTRACT TRUNCATED AT 250 WORDS)

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

A total of 17 couples with repetitive implantation failure after transfer of fresh or frozen-thawed embryos had half of their zygotes cultured in standard conditions and frozen at day 2 after insemination, and the other half cocultured with autologous granulosa cells and transferred at the morula or blastocyst stage at day 5 or 6 after oocyte retrieval. At the end of the culture period, supernatants of cocultures were recovered for steroid assays. Monolayers were stained for granulosa cell growth and morphological assessment. We observed that granulosa cells improve embryo development in vitro since 32 out of 60 (53%) reached the morula stage and 18 (30%) the blastocyst stage, leading to a total of 83% embryos available for transfer (compared with 3% without coculture). The ongoing pregnancy rate of these patients who were selected because they had at least three previous implantation failures, is only 5.9%, however, which is similar to the control group without coculture (6.3%). To conclude, granulosa cells improve embryo development but not the pregnancy rate after transfer of cocultured embryos in patients with multiple previous implantation failures.

Ten couples included in our in-vitro fertilization programme, selected because of one of more previous total fertilization failures or a low fertilization rate (20%), were entered in an experimental protocol of egg insemination on autologous granulosa cells. Half the oocytes from each patient were randomly assigned to either a control or a co-culture group. We observed no difference in the fertilization rate between the control (16.2%) and co-culture groups (12.1%). Only two couples benefited from this technique since fertilization was obtained only in co-culture. The poor efficiency of this protocol led us to propose the use of sperm micro-injection if all classical attempts to improve fertilization were unsuccessful.

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.

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 patient's 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.

More than 60% of patients with polycystic ovary disease (PCO) cannot conceive after repeated ovulation inductions with Clomifene citrate although there is ovulation or more frequently follicle luteinization. Because of hyperstimulation, therapy with hMG has been superseded by low doses of purified FSH with variable results according to authors. It has been even claimed that there was no benefit to replace hMG with FSH. However, on the basis of the PCO physico-pathology, namely LH hypersecretion and androgen hyperproduction, it would be rational to associate the desensitization of the pituitary with LH-RH agonist and the ovary stimulation with variable doses of hMG or purified FSH. In the series where such therapy associating LH-RH agonists with purified FSH was applied, the results concerning suppression of LH and androgen secretion, and the occurrence of pregnancy were interesting. However, the risk of hyperstimulation still occurred. Thus, the first part concerns the critical review of these results while, in the second part, our experience in in vitro fecundation will be reported.

The success rate of human embryo cryopreservation depends on technical and embryonic parameters. First of all, the cryoprotectant can affect embryo survival as we found by comparing two freeze-thaw procedures using propanediol (PROH) (1.5 mol) alone or with sucrose (0.1 mol). Embryo survival was significantly enhanced with sucrose (62 versus 32%). Embryo quality is another major parameter involved in the success of freezing; the rates of positive survival were found to be 67% for morphologically normal embryos versus 49% for embryos with fragments (P less than 0.001). The efficiency of embryo cryopreservation in an IVF programme could be estimated in 1986: a woman with extra embryos, stored after transfer of 3-4 fresh embryos (16% of all cycles), can expect a 22% pregnancy rate per transfer of fresh embryos and a 32% pregnancy rate per collection after transfer of the stored eggs. A comparative study of the cryopreservability of immature or mature oocytes was performed in humans. Human oocytes have a low survival rate (36%) whatever the cryopreservation protocol or the initial maturation stage. Immature human oocytes could survive freezing and thawing, mature and be fertilized in vitro, but with a very low efficiency.

The best moment for human embryo transfer has not yet been accurately determined. The human uterus is able to receive young embryos (2, 4 cell-stage and even pronucleated eggs), insure their growth and implantation at a rate that does not exceed, however, 15% for one transferred egg. At the present time in vitro culture to the blastocyst stage, which impairs human embryo viability, should be avoided. Contrary to classical IVF, it becomes possible to dissociate embryo and endometrial ages when transferring frozen-thawed eggs. Our study concerns 443 spontaneous, stimulated or artificial cycles, performed in patients with normal or without endogenous ovarian function (tabl. 1). There was trend towards enhanced pregnancy rates (17%) for synchronous as compared to one-day asynchronous transfers (9%) (tabl. 2). Similar data, widely stated in mammals, emphasize the necessity of a precise chronology of embryo transfer. Embryos only survive and get implanted when placed in a receptive uterus. The onset of the refractory period appears to be closely linked to the maternal steroid environment. Consequently, no pregnancies arose from transfer of donated embryos beyond 6 days of progestative supply in women deprived of endogenous ovarian function. In the same way, administration of progesterone 40 h before oocyte recovery seemed to advance the refractory phase. Indeed, a reduction in the pregnancy rate per transfer was observed in such circumstances without any obvious impairment of embryo viability (tabl. 4). The best pregnancy rate was obtained in synchronous transfers while a one-day disynchronization reduced this rate by half.

Freezing and thawing (F - T) was applied to 490 early human embryos using propanediol as cryoprotectant. The survival rate of embryos frozen with propanediol alone did not exceed 31% (26/83). The combination of propanediol and sucrose, however, significantly increased the percentage of surviving (248/407 = 61%) and intact (188/407 = 46%) embryos and seemed to enhance embryo viability as suggested by the implantation rate (14.5 versus 8%) without, however, any statistical significance. Embryo survival, but not viability, was correlated with morphological features, whereas neither the age of embryos (1, 2 or 3 days post-insemination) nor the segmentation stage (regular or intermediate) were involved in F - T ability. Thirty-eight F - T embryos implanted when replaced in utero, representing 8% of all F - T embryos and 14% of the F - T replaced embryos. The pregnancy rate per transfer reached 19% (35/185) and was identical to the pregnancy rate per transfer of fresh embryos (253/1149 = 22%). In oocyte donation, too, embryo freezing did not impair the pregnancy rate (25%). In spontaneous cycles, synchronous transfer gave better results than asynchronous transfers (20 versus 10%), but spontaneous cycles had no significant advantage (16% pregnancy/transfer) as compared to stimulated (26%) and artificial (27%) cycles.

Fertilization in-vitro offers the possibility of studying the karyotype of ovocytes obtained after superovulation, when they are not fertilized. Among 120 ovocytes, 30 p. cent presented a chromosomal anomaly. The same study was carried out on morphologically normal or abnormal embryos - and the percentage of chromosomal anomalies approximates here 27 p. cent. These studies offer a model of natural selection against chromosomal anomalies and confirm the limiting role of these anomalies in the success of FIV.

In vivo, the normal regulation of embryo growth passes by a complex system of signals exchanged between the ovary (corpus luteum, CL), the genital tract, and embryo itself. So failure may result from various abnormalities in and between these three “partners.” Current knowledge regarding early embryo development, is only partial, and improvement techniques are limited if not nonexistent; in various species, viability quickly declines and cleavage stops around the so-called block stage.

193 patients had 212 cycles induced for in vitro fertilisation between May 1983 and May 1984. 20 pregnancies of which 10 are still in progress were obtained with 6 spontaneous abortions. There were 2 so-called biochemical pregnancies and 3 extra-uterine pregnancies. The figure of 17% pregnancies where embryos were replaced totals less than 10% if one considers those pregnancies that really carry on. The number of extra-uterine pregnancies is disturbing. Finally, 2 multiple pregnancies continued, one of them being a quadruple pregnancy. The authors have made a special study to see the parameters of the conditions under which the pregnancies were obtained and the prognosis for such pregnancies.

Abnormal embryo development represents the major cause of implantation failures and accounts for the low rate of human fertility in vivo or in vitro. Chromosome abnormalities are widely involved in this process as 26% of oocytes, 8% of fertilizing spermatozoa and 29% of preimplantation embryos carry a chromosome aberration induced by meiotic (aneuploidy) or mitotic (mosaic) non disjunctions. Fertilization anomalies (possibly increased by in vitro procedures) were recorded: 1.6% of embryos resulted from parthenogenesis and 6.4% were polyploid (mainly polyspermic). A morphological, histological and ultrastructural study of embryos recovered after in vivo or in vitro fertilization showed some anomalies: multinucleated blastomeres, cytoplasmic fragments in the perivitelline space, vacuoles, associated or not with developmental impairement. Finally, a few embryos appeared to be free of abnormalities. The analysis of in vitro developmental capacities of normal or abnormal embryos showed great differences: parthenones exceptionally reached the blastocyst stage and therefore probably did not implant. The diploid embryos used in this study were (for ethical reasons) more or less fragmented and gave evidence of low developmental capacities, limited to the 3rd cleavage. Triploid embryos were able to further develop as some of them reached the early blastocyst stage; they represented the major cause of chromosomal 1st trimester abortions. It is interesting to note that 47% of tripronucleated ova divided directly into 3 and 6 cells (probably via a tripolar spindle) instead of 2 and 4 cells as classically described. Finally, tetraploid embryos expressed a precocious lethality as none developed beyond the 3rd cleavage. To conclude, many embryos carry genetic and/or cytological abnormalities which may be enhanced by superovulation treatments. The selection proceeds through all pre- and postimplantation steps, and as a matter of fact nor more than 0.6% newborns are abnormal.

More than 60% of patients with polycystic ovary disease (PCO) cannot conceive after repeated ovulation inductions with Clomifene citrate although there is ovulation or more frequently follicle luteinization. Because of hyperstimulation, therapy with hMG has been superseded by low doses of purified FSH with variable results according to authors. It has been even claimed that there was no benefit to replace hMG with FSH. However, on the basis of the PCO physico-pathology, namely LH hypersecretion and androgen hyperproduction, it would be rational to associate the desensitization of the pituitary with LH-RH agonist and the ovary stimulation with variable doses of hMG or purified FSH. In the series where such therapy associating LH-RH agonists with purified FSH was applied, the results concerning suppression of LH and androgen secretion, and the occurrence of pregnancy were interesting. However, the risk of hyperstimulation still occurred. Thus, the first part concerns the critical review of these results while, in the second part, our experience in in vitro fecundation will be reported.