Factors involved in the success of human embryo freezing. Does cryopreservation really improve the IVF results?
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Embryo Transfer↗
Biomedical subjects
J Mandelbaum
Publications and source records attributed to J Mandelbaum.
[Timing of embryo transfer and success of pregnancy in the human].
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.
Animals↗
[Failure of embryonic development].
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.
Chromosome Aberrations↗
[Evaluation criteria of the condition of the endometrium in relation to the presence of ovocytes].
18 patients with ovarian failure received cyclic steroid replacement therapy including estrogens and progesterone for embryo transfer. Hormonal results and endometrial histology are reported. The temporal window of endometrial receptivity 13 discussed, shedding some light on the respective role of embryo and endometrium in the implantation process.
Administration, Intravaginal↗
[Chromosome analysis of ovocytes and human embryos collected after fertilization in vitro. A model of natural selection against aneuploidy].
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.
Aneuploidy↗
[Solutions provided by the freezing of embryos and questions posed by the freezing of human ovocytes].
The major questions raised by frozen embryos are reviewed: how to freeze? which embryos to freeze? how to replace the frozen embryos? What is he contribution of frozen embryos in a program of fertilization in-vitro. The cryopreservation of human ovocytes is also discussed.
Embryo Transfer↗
Chromosome investigations in early life. II. Human preimplantation embryos.
Cytogenetic analysis of 68 human embryos at the 2- to 8-cell stage was performed according to Tarkowski's technique. Sixteen per cent of diploid embryos showed abnormalities, essentially diploid/haploid or triploid/haploid mosaicism. Considering the aspect of the embryos, 11% of healthy looking and 19% of fragmented embryos were chromosomally abnormal without, however, any statistical significance in this small series. Only 46.7% of the tripronucleate fertilized eggs showed a triploid chromosome complement. In 20% of the cases, however, diploid metaphases were found, and in the last 30% a triploid/diploid mosaicism. One per cent of the oocytes displayed a single pronucleus, and the resulting embryos contained haploid sets of chromosomes suggesting a parthogenetic activation. The overall rate of chromosome abnormalities, including 16% of abnormal diploid eggs, 6% of polyploid and 1% of haploid embryos, thus reaches 23% in this series.
Aneuploidy↗
Ultrastructural and autoradiographic observations on multinucleated blastomeres of human cleaving embryos obtained by in-vitro fertilization.
Human embryos from the 2-cell to the morula stage developing in vitro after monospermic fertilization were incubated with [3H]thymidine or [3H]uridine and those possessing multinucleated blastomeres were examined by conventional transmission electron microscopy and by light- and electron-microscope autoradiography. Labelled DNA was present in all nuclei showing normal ultrastructural appearance, in pseudonuclei lacking nucleoli and their precursors and often demonstrating an unusual chromatin organization, free in the cytoplasm in structures resembling aggregates of dense chromatin and in small cytoplasmic vesicles in the close vicinity of these aggregates. The labelling with [3H]thymidine was not detected in about 50% of the cytoplasmic chromatin aggregates, suggesting that this extranuclear DNA was no longer replicated. Signs of extrusion of pseudonuclei to the extracellular space were occasionally observed. RNA synthesis could not be detected on free cytoplasmic chromatin, while [3H]uridine was incorporated into all nuclei and most pseudonuclei of multinucleated blastomeres of embryos at the 8-cell and morula stages. However, the major outburst of transcriptional activity and the ultrastructural cytoplasmic changes typical of the assumption of embryonic genome expression, occurring normally at the 8-cell stage, were observed only exceptionally.
Autoradiography↗
Human embryo cryopreservation, extrinsic and intrinsic parameters of success.
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.
Blastomeres↗
High-resolution autoradiographic localization of DNA-containing sites and RNA synthesis in developing nucleoli of human preimplantation embryos: a new concept of embryonic nucleologenesis.
Human embryos from the 2-cell to the morula stage, obtained by in vitro fertilization, were incubated with [3H]thymidine or [3H]uridine so as to achieve labelling of all replicating nuclear DNA and the newly synthesized RNA, respectively. The label was localized in different structural components of developing nucleoli using electron microscopic autoradiography. Careful study of the relationship between the structural pattern and nucleic acid distribution made it possible to define four stages of embryonic nucleologenesis. Homogeneous nuclear precursors (i) consist of nucleolar matrix elements appearing as filaments of 3 nm thickness, (ii) do not contain recently replicated DNA and (iii) lack RNA synthetic activity. Penetration of DNA into these bodies is a key event leading to their transformation into heterogeneous nucleolar precursors. In addition to the 3 nm matrix filaments, two types of 5 nm fibrillar components can be recognized in them. The denser type contains DNA and is the site of nucleolar RNA synthesis, while the more loosely arranged 5 nm fibrils are not labelled with [3H]thymidine and apparently represent the newly produced pre-rRNA detached from the transcribing rDNA filament. Compact fibrillogranular nucleoli are characterized by the first appearance of the granular component and reduction of the nontranscribing part of the fibrillar component, both indicating the activation of the machinery for rRNA processing. Finally, the granular component is most evident in reticulated nucleoli, occupying mostly the inner parts of their nucleolonema, while the transcription sites tend to be located at the nucleolar periphery. Our findings advocate a unique concept of embryonic nucleologenesis, different from any other nucleolar event during the cell cycle of differentiated cells. This developmental pattern is characterized by a gradual activation of rRNA synthesis and processing, mediated by progressive association of rDNA and, later on, the newly formed pre-rRNA with pre-existing nucleolar matrix elements that are originally topically separated from nucleolar organizer regions. This model may have a general validity in early animal embryos despite some interspecies variability in the timing of individual steps and resulting structural peculiarities.
Autoradiography↗
[Pregnancies after freezing of human ova at Tenon Hospital].
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Embryo Transfer↗
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.
Aneuploidy↗
Ultrastructure of the human preovulatory oocyte.
The ultrastructure of preovulatory human oocyte-cumulus complexes was described after inducing maturation by clomiphene, human menopausal gonadotropin (hMG), human chorionic gonadotropin (hCG) treatment. The majority of the oocytes was at metaphase II of meiosis, with a radially orientated spindle. The oocyte surface was covered by a multitude of microvilli. Cortical granules were nonuniformly distributed along the cortex. A cytoplasmic polarization was observed. The cytoplasmic organelles were in general uniformly dispersed, with the exception of a narrow segment within which cytoplasmic membranes and mitochondria formed clusters. The spindle was usually found at the borderline between the two regions of the cytoplasm. The functional significance of this polarization is not yet known.
Chorionic Gonadotropin↗
Nucleologenesis in the human embryo developing in vitro: ultrastructural and autoradiographic analysis.
Nucleolar development during cleavage of human embryos in vitro was analyzed using combined autoradiographic and ultrastructural approaches. Human 2- to 4-cell embryos lack functionally active nucleoli. They possess nucleolus-like bodies formed by a homogeneous mass composed of densely packed fine fibrils. The nucleolus-like bodies contain no recently replicated embryonic DNA and do not show any detectable RNA synthesis. Their transformation into nucleoli is a relatively rapid process characterized by progressive infiltration of these bodies by adjacent chromatin, followed by the beginning of RNA synthesis and processing reflected by the appearance of the first nucleolar granules. These changes take place in 6- to 8-cell embryos, probably starting after the third cleavage division. Finally, most frequently in 10- to 12-cell embryos, typical nucleolar structure is established as a result of intranucleolar differentiation giving rise to distinct fibrillar and granular components as well as to nucleolar interstices. It is suggested that this pattern of nucleologenesis, different from that pertinent to mouse embryos (M. Geuskens and H. Alexandre (1984). Cell Differ. 14, 125-134), might be related to the relatively late activation of embryonic rRNA genes transcription and to the expressed rapidity of this process.
Blastomeres↗
Steroid profiles during cycles with HCG stimulation of luteal function during in-vitro fertilization cycles.
178 in-vitro fertilization cycles have been explored for luteal function. Clomiphene and human menopausal gonadotrophins were used for ovarian stimulation and some patients were supplemented with human chorionic gonadotrophin (HCG) (5000 IU) on the day of laparoscopy, and also 2 and 4 days later. An imbalance of luteal function was observed in non-supplemented stimulated cycles, with hyperoestrogenicity and a relatively low production of progesterone. The administration of HCG reduced this imbalance by improving the production of progesterone without affecting the production of oestradiol-17 beta.
Adult↗
Timing of in-vitro fertilization of cumulus-free and cumulus-enclosed human oocytes.
In humans, in contrast to other species, sperm capacitation requires a very short time, as in-vitro fertilization has been obtained after only 45 min of contact between oocytes and spermatozoa capacitated for 1 h. No fertilization occurred, whatever the duration of sperm capacitation, when gamete mixing did not exceed 30 min. On the contrary, 85% of cumulus-free mature oocytes exposed to sperm for 1-4 h were fertilized. The presence of the pre-ovulatory, fully-expanded or compact cumulus mass did not represent a physical barrier to sperm progression, as we observed no delay in fertilization when oocytes were enclosed in the cumulus. The use of a short insemination protocol (1-4 h instead of 17-20 h) did not reduce the fertilization rate of denuded or cumulus-enclosed oocytes and had no significant effect on the morphological appearance of the embryos or their cleavage rates.
Fertilization in Vitro↗
Chromosome investigations in early life. I. Human oocytes recovered in an IVF programme.
Fifty-five oocytes recovered in an in-vitro fertilization (IVF) programme and remaining unfertilized when observed 42 h after insemination were prepared for chromosomal analysis. Sixteen oocytes displayed no polar body at the time of fixation and were supposed to be in metaphase I. In fact only two of them were in diakinesis, the others containing a diploid set of metaphase II chromosomes indicating that in 89% of the cases oocytes achieved meiosis without any extrusion of the first polar body. Thirty-nine oocytes in metaphase II were analysed. Nine were abnormal showing three D nullosomies, one G disomy, one double disomy for a 3 and a D chromosome, one deletion of the long arm of a G chromosome, one cell with extra chromosomes and/or chromosome breaks, one endoreduplication and one tetraploidy. The overall rate of abnormalities reached 22%. This high rate of chromosome anomalies can be explained by the nature of this population of fertilization failure, the frequently advanced maternal age and the use of superovulation treatments.
Chromosome Aberrations↗
Activation of nucleolar and extranucleolar RNA synthesis and changes in the ribosomal content of human embryos developing in vitro.
RNA synthetic activity of human 2-16-cell embryos developing in vitro was studied by [3H]uridine light-microscope autoradiography. Parallelly cut thin sections were examined in the electron microscope. The first extranucleolar RNA synthesis was detected in 4-cell embryos, but nucleoli were never labelled until the 3rd cleavage (6-8-cell embryos). In 6-cell embryos the nucleolar labelling was mostly confined to a narrow peripheral zone. In later cleavage stages most of the blastomeres showed intensive labelling of nucleoli and extranucleolar chromatin. However, rather low levels of extranucleolar RNA synthesis and the absence of nucleolar activity were often seen even in blastomeres of fully compacted morulae. The activation of nucleolar RNA synthesis entailed a noticeable increase in the number of ribosomes (estimated by electron microscope morphometry) that followed a marked drop during the period between the 2-cell and 8-cell stages. The results indicate that the concentration of ribosomes in the preovulatory oocyte is a major factor of its developmental potential.
Autoradiography↗