Michelle Plachot

Early morphological signs of embryonic genome expression in human preimplantation development as revealed by quantitative electron microscopy

A quantitative electron microscopic analysis of human preimplantation embryos in conjunction with [3H]uridine labeling and light microscopic autoradiography revealed significant differences in the fractional volume of some cell organelles between the blastomeres of eight-cell embryos with fully activated extranucleolar and nucleolar transcription and those showing low extranucleolar and no nucleolar RNA synthesis, a pattern typical of four-cell human embryos. The latter type of blastomeres in eight-cell embryos did not show any significant quantitative cytological difference when compared to blastomeres of four-cell embryos. The phenotypical changes accompanying the overall enhancement of the embryonic transcriptional activity (increase in tubules/vesicles ratio and lysosomes, decrease in Golgi apparatus) were due to repartition of intracellular membranes amongst different types of organelles rather than to a noticeable change in the existing equilibrium between total membrane production and degradation.

Incidence of sex chromosome abnormalities in spermatozoa from patients entering an IVF or ICSI protocol

OBJECTIVE The objective of this study was the determination of sex chromosome aneuploidy frequency in spermatozoa from patients included in an in vitro fertilization (IVF) or intra cytoplasmic sperm injection (ICSI) protocol. METHODS Spermatozoa from nineteen patients, including patients with normal seminal parameters according to World Health Organization (WHO) criteria and patients exhibiting abnormal seminal parameters, were analyzed by dual color fluorescence in situ hybridization (FISH) for aneuploidies of the X and Y chromosomes. Our technique, using only probes for sex chromosomes and not for autosomes, does not discriminate between hyperhaploid and diploid sperm nuclei. The results were analyzed in two different ways: in relation to the semen status, denoted normal or abnormal and with regard to the ability of the sperm to fertilize oocytes when IVF or ICSI was performed. RESULTS Abnormal semen showed a significant increase in the overall rate of sperm nuclei with XY, XX and YY sex chromosome complements, 1.59% compared to normal semen, 0.78% (p<0.02). Semen shown to be able to fertilize oocytes only by ICSI showed a higher incidence of XY-bearing spermatozoa, 1.26%, compared to semen able to fertilize oocytes by conventional IVF, 0.37% (p<0.001). The incidence of XX-or YY-bearing sperm nuclei was also significantly elevated in the ICSI group (0.25% XX, 0.50% YY) (p<0.02) as compared to the IVF group (0.06% XX, 0.16% YY). CONCLUSIONS We concluded that infertile men requiring ICSI treatment showed a higher incidence of sex chromosome aneuploidy, due to meiosis I and II nondisjunction, in their spermatozoa as compared to men requiring IVF for reasons of predominantly female infertility.

Sequential use of clomiphene citrate, human menopausal gonadotropin, and human chorionic gonadotropin in human in vitro fertilization. II. Study of luteal phase adequacy following aspiration of the preovulatory follicles*

The 88 patients included in the in vitro fertilization program during 113 cycles were submitted to superovulation by sequential use of clomiphene citrate, human menopausal gonadotropin, and human chorionic gonadotropin. No correlation was found between estradiol and progesterone levels during the luteal phase and estradiol on the days preceding administration of human chorionic gonadotropin. Nineteen biopsies of the endometrium were carried out. The importance of the increase of estradiol between the day before and the day of administration of human chorionic gonadotropin is positively correlated with the quality of the endometrium.

Ultrastructure of the human preovulatory oocyte

The ultrastructure of preovulatory human oocyte—cumulus complexes was described after inducing maturation by clomiphene, human menopousal 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 spirndle was usually found at the borderline between the two regions of the cytoplasm. The functional significance of this polarization is not yet known.

CHROMOSOME ANALYSIS OF OOCYTES AND EMBRYOS

As an extension of IVF techniques, preimplantation genetic diagnosis will make it possible to detect genetic and chromosomal diseases in embryos. With the use of molecular genetic technology, such as Polymerase Chain Reaction (PCR), several gene mutations have already been detected in polar bodies aspirated from mature oocytes or in blastomeres from biopsied embryos (Verlinsky, this volume). The same approach was used for gender determination in cases at high risk of X-linked diseases to avoid the birth of affected males (Handyside, this volume; Milayeva, this volume).

Programmed ovulation induction and oocyte retrieval for in vitro fertilization

Forty-two patients underwent programmed ovulation induction for oocyte retrieval. They were treated in the preceding cycles with a progestagen, ethynodiol diacetate, at a dose of 2 mg twice daily. Two groups were defined based upon the stimulation protocol: Group A1 was stimulated with clomiphene citrate and human menopausal gonadotropin (hMG), and Group A2 with follicle-stimulating hormone (FSH) and hMG. They were compared to two randomized control groups of patients who received the same induction but were classically monitored. There was a high proportion of spontaneous ovulations in the programmed group (8/42) compared to the nonprogrammed group (0/42). There was a nonsignificant difference in the number of oocytes obtained or embryos replaced per cycle. Four pregnancies were obtained in the programmed group (24% per transfer), against 10 in the nonprogrammed patients (32% per transfer). The results of this method seem to be better using FSH for ovulation stimulation and a verification of the serum estradiol on the day of induction with human chorionic gonadotropin (hCG) and the following day (semiprogrammed method).

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.

Regulations for preimplantation genetic diagnosis in France.

A commentary on regulations for PGD in France.

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.