Sperm-zona binding and sperm-oocyte penetration analysed by DNA fluorescence: a test for gamete quality after in-vitro fertilization.

Sperm-zona binding, sperm-oocyte penetration and pronucleus formation in oocytes obtained after in-vitro fertilization (IVF) failure were studied by analysing DNA fluorescence. Oocytes were classified according to their maturational status, sperm-zona pellucida binding and sperm-oocyte penetration. Spermatozoa were classified as normal or abnormal according to conventional parameters. Sperm-zona binding and sperm-oocyte penetration into uncleaved oocytes were positively related to fertilization: 6.2% of those oocytes from cohorts where none of the oocytes fertilized had > 10 spermatozoa bound to the zona, and 7.7% of them were penetrated. When at least one oocyte per cohort fertilized, these rates were 37.1 and 41.1% respectively. Sperm-zona binding is related more strongly to sperm-oocyte penetration, even without pronucleus formation, than to the maturational status of the oocyte: 26.9% of the oocytes were penetrated by spermatozoa, of which 20.7% had reached the pronuclear stage. For 25 out of 78 couples with a total IVF failure, whatever the spermiogram, defective sperm-zona binding, sperm-oocyte penetration or pronucleus formation was observed. Sperm-zona binding and penetration were positively related, especially when oocytes came from patients with partial IVF failure. Clinical information concerning the fertilizing capacity of oocytes and spermatozoa after IVF failure can be obtained by the simultaneous analysis of sperm-zona binding and sperm-oocyte penetration, and whether or not the latter results in pronucleus formation.

[Stress and premature menopause]. / Stress et ménopause précoce.

By acting on the neuroendocrine system during the stress response, life events may greatly affect homeostasis and favor the appearance of disease. Here, we describe a relationship between stressful life events and premature ovarian failure. From a mechanistic point of view, we suggest an autoimmune origin for such premature ovarian failure on the basis of the role of cytokines in folliculogenesis and of their increased production during stressful life events.

Timing of pronuclear development and first cleavages in human embryos after subzonal insemination: influence of sperm phenotype.

The sequential transformations of human sperm nuclei in human eggs after subzonal insemination (SUZI; n = 104) and the influence of sperm defects on this timing were studied. This chronology was compared to that of two control series of zygotes obtained after SUZI with normal spermatozoa (n = 35) and after in-vitro fertilization (IVF) with normal donor spermatozoa (D-IVF; n = 220). Pronuclear formation took place between 4.5 and 10.5 h post-SUZI for 92.8% of the zygotes. They remained visible for 13 h and began to disappear 18.5 h post-SUZI. The timespan 3 h. Zygotes obtained after D-IVF had a similar rate of pronuclear disappearance but approximately 4 h later. The second cell cycle was more rapid for zygotes obtained by D-IVF than by SUZI, but the developmental rate of zygotes obtained by SUZI varied according to sperm phenotypes. For patients with previous unexplained IVF failures (control group with normal spermatozoa), the developmental rate was lower, suggesting the influence of oocyte quality. In conclusion, the end of the first cell cycle of zygotes obtained by insemination under the zona pellucida appears 4 h earlier compared to zygotes obtained after insemination outside the zona pellucida.

In vitro DNA fluorescence after in vitro fertilization (IVF) failure.

PURPOSE: A pilot study was performed to test the diagnostic value of in vitro DNA fluorescence in oocytes that failed to fertilize after IVF. Ten patients with a cleavage rate less than 20% after IVF were included. RESULTS: Uncleaved oocytes were observed by fluorescence microscopy after incubation with the DNA fluorescent dye Hoeschst 33342. Four main causes which may have contributed to the low cleavage rate were found: (1) sperm incapacity to penetrate the oocyte despite the absence of the usual criteria for male infertility, (2) oocyte immaturity, (3) delayed fertilization, and (4) oocyte abnormalities revealed by aberrations in the morphology of the female chromatin. CONCLUSIONS: The possibility of a rapid and detailed analysis of the maturational status of unfertilized oocytes, the morphology of the female chromatin, the presence and quantity of spermatozoa tightly bound to the zona pellucida, and sperm penetration into the oocyte without subsequent pronucleus formation, using DNA fluorescence, allows us to clarify further the cause of fertilization failure and to orient infertility treatment toward the male, the female, or both partners.

Fertilization and ageing processes in non-divided human oocytes after GnRHa treatment: an analysis of individual oocytes.

Some human oocytes cultured together with spermatozoa for in-vitro fertilization (IVF) do not subsequently divide. The arrest of the fertilization process at different moments during development may provide information about the cause of fertilization failure. Oocytes which subsequently divide are transferred 48 h after insemination; when oocytes do not divide, ageing processes can be observed. Therefore these oocytes are interesting material in which to observe both fertilization and ageing. Our study concerns 72 undivided human oocytes 0, 48 or 72 h post-insemination. DNA of the oocyte and spermatozoa was visualized by the DNA fluorescent dye Hoechst 33342. Living oocytes were observed in toto by fluorescence and bright field microscopy which allowed nuclear and pronuclear membranes to be discerned. Oocytes were subsequently fixed and sectioned for bright field microscopy. Both techniques allowed parallel observations. Oocytes at various stages of fertilization are described: sperm penetration in both mature and immature oocytes, decondensation of sperm-heads, premature condensation of male chromatin, polyspermy and pronucleus formation. Typical ageing processes such as the centripetal migration of the metaphase II chromosomes, the formation of a restitution nucleus and the lagging of chromosomes within a metaphase spindle are observed. DNA fluorescence appears to be a quick, easy and valuable means to analyse fertilization and its failure.

Effects of varying doses of HCG on the evolution of preovulatory rabbit follicles and oocytes.

To determine the effects of insufficient or excessive doses of human chorionic gonadotrophin (HCG) on ovulation, varying single doses from 5 to 100 IU were administered to 40 does in oestrus. A total of 371 preovulatory follicles and oocytes were observed. Low doses of HCG (5-10) started resumption of meiosis, but no ovulation occurred. Higher doses progressively induced nuclear maturation (prometaphase, metaphase I, metaphase II). Simultaneously increasing doses initiated ovulation of some of the follicles, then of most of the follicles. Unruptured follicles, mostly with oocytes in metaphase II were frequent and depending on the dose, these were preovulatory, haemorrhagic or luteinized. The administration of different doses demonstrated the possible dissociation of several mechanisms leading to ovulation. The induction of nuclear maturation requires lower doses of HCG than luteinization. Follicular rupture requires even higher doses. Premature luteinization induces intrafollicular ovum retention without (LUF syndrome) or with follicular rupture. These mechanisms of ovulation explain the effects of blunted luteinizing hormone surges or inadequate HCG administration.

Oocyte size and intrafollicular position in polyovular follicles in rabbits.

Healthy follicles with 2-24 oocytes were observed in adult rabbit ovaries during all phases of folliculogenesis from primary to preovulatory follicles. Most follicles contained 2-3 oocytes which developed according to their topographical situation in the follicle. The central oocyte in a normal topographical situation has an almost normal growth and development up to metaphase II and cumulus expansion. The peripheral oocytes grow more slowly: most do not attain the normal size or resume meiosis and remain surrounded by ordinary granulosa cells. When the number of oocytes is higher than 3, the peripheral oocytes develop even more slowly, as do the central ones. It demonstrates the necessity for the oocyte to occupy a certain position inside the follicle and to reach a size which allows resumption of meiosis; the cumulus responds only to oocytes of normal size and position. We suggest that, despite the relative frequency of binovular follicles, fertilization of two oocytes originating from one follicle is unlikely.

Mature and immature oocytes in large and medium follicles after clomiphene citrate and human menopausal gonadotropin stimulation without human chorionic gonadotropin.

After ovarian stimulation with clomiphene citrate combined with human menopausal gonadotropin, 24 large (greater than 16 mm) and 16 medium (7 to 15 mm) human follicles were classified according to plasmatic estradiol (E2), luteinizing hormone (LH), and histology of the follicle and oocytes (in thin sections). An asynchronism of several hours between the stage of development of the largest and large cohort follicles is observed; overripeness of oocyte cumulus complex (OCC) is revealed. An asynchronous response to gonadotropins in granulosa and cumulus is also seen in the cohort of medium follicles of the same ovary, but not resumption of nuclear maturation of the oocyte. The efficiency of these oocytes after fertilization is discussed.

Timing of nuclear maturation and cumulus dissociation in human oocytes stimulated with clomiphene citrate, human menopausal gonadotropin, and human chorionic gonadotropin.

The maturation of the preovulatory human follicle larger than 16 mm is described between 0 and 36 hours after human chorionic gonadotropin (hCG) administration. The nuclear evolution of the oocyte is timed: prophase at 14 hours, germinal vesicle breakdown at 15 hours, metaphase I at 20 hours; metaphase II is complete by 35 hours. The quantified expansion of the cumulus is related to nuclear evolution. Almost complete expansion may already be observed 18 hours before ovulation (20 hours after hCG). The specific action of clomiphene citrate, human menopausal gonadotropin, and hCG stimulation on the granulosa induces a more rapid luteinization than is found after other means of stimulation or in spontaneous cycles. Oocytes from follicles less than or equal to 16 mm on preovulatory ovaries often show a reaction to hCG that may be related to their subsequent quality.

Relationship between the characteristics of the menstrual cycle and congenital malformations in the human.

We have studied the characteristics of conceptional and pre-conceptional cycles in mothers of malformed infants. A comparison made with a control group of mothers of normal, term infants showed that for the former the hypothermic phase during the conceptional cycle was longer than for the latter (20.4 and 16.9 days, respectively). Moreover, the mothers of malformed infants showed a slower temperature rise (greater than 3 days) in 45% of cases, vs 28% in the controls. The menarche of the mothers of malformed infants occurs later (14.3 vs 12.8 years). They usually have long menstrual cycles and a bad obstetric and gynaecological history. The risk for congenital malformations is thus closely related to the length of the hypothermic phase and to a slow temperature rise in the conceptional cycle. Therefore we suggest that the preovulatory oocyte overripeness is one of the mechanisms of congenital malformation.

Delayed ovulation and monozygotic twinning.

Aiming to show that delayed ovulation may induce MZ twinning, follicular maturation was induced in the rabbit by a small quantity of Pregnant Mare Serum Gonadotropin (16 UI four times): coitus, which induces ovulation in the rabbit, was delayed 60 hours after the last injection. From the 387 blastocysts obtained after this treatment, 6 (1.5%) were pairs of MZ twins, twinning being otherwise exceptional in the rabbit. Other anomalies were shown by the embryos, apparently related to a deficient quality of the eggs: high embryonic mortality (62% vs. 27% in controls) and chromosomal anomalies (20%) such as trisomies, triploidies, and chimaeras. The relation between MZ twinning, chromosomal anomalies, and embryonic mortality induced by delayed ovulation, could be connected and related to the poor perinatal conditions frequently observed in human MZ twins.