Concerted action of human chorionic gonadotropin and norepinephrine on intracellular-free calcium in human granulosa-lutein cells: evidence for the presence of a functional alpha-adrenergic receptor.

Luteal cells are known to possess receptors for LH/hCG and receptors of the beta-adrenergic type. Interactions of specific agonists with either receptor lead to the activation of adenylate cyclase and subsequently to an increase of cAMP. Since in the human there is also evidence for the presence of alpha-adrenergic receptors, we have investigated whether activation of these receptors is linked to calcium as a second messenger and performed measurement of intracellular free calcium (Ca2+) with Fura-2 in single human granulosa-lutein cells. Addition of either hCG (100, 1,000, 25,000 IU/L) or norepinephrine (NE; known to interact with both alpha- and beta-adrenergic receptors), beta-adrenergic receptor agonist isoproterenol (ISO), or alpha-adrenergic receptor agonist phenylephrine (PHE; all at 10 and 100 mumol/L) did not increase free intracellular Ca2+. However, the addition of combinations of NE/hCG, PHE/hCG, but not the combination ISO/hCG, induced a transient increase in cytosolic free Ca2+. The NE/hCG-evoked calcium signal was not abolished in the presence of the beta-adrenergic receptor antagonist propranolol and was not affected by removal of extracellular Ca2+. Furthermore, we tested whether catecholamines affected the release of progesterone in the presence or absence of hCG. As expected, hCG (10,000 IU/L) stimulated progesterone release by cultured granulosa-lutein cells. When these cells were incubated with NE, PHE, or ISO (at 10 mumol/L), production of progesterone by these cells was not affected. However, the combinations of NE and PHE with hCG abolished the hCG-induced progesterone accumulation, but ISO coincubated with hCG did not. Taken together, our results indicate: 1) the presence of functional alpha-adrenergic receptors on human granulosa-lutein cells; 2) simultaneous activation of two different receptors (for hCG and alpha-agonists) are able to evoke intracellular Ca2+ elevation, implicating postreceptor interactions in human granulosa lutein cells; 3) this process occurs even in the absence of extracellular Ca2+, indicating the involvement of intracellular Ca2+ stores, most likely due to activation of phosphoinositide pathway; 4) catecholamines most likely acting via alpha-adrenergic receptors, inhibit the LH/hCG-induced release of progesterone.

Sperm chromosomes and habitual abortion.

Genetic factors, especially numerical chromosome anomalies, play an important role in embryonic loss. Because somatic cell analysis cannot assess the risk of errors arising de novo during germ cell maturation, we investigated whether the male gametes from couples with habitual abortion carry a higher rate of anomalies than those from donors without reproductive dysfunction. Our results indicate that there is no significant difference between the two groups for the total rates of aneuploidy and structural anomalies. However, the levels of chromosome breaks and acentric fragments were significantly higher in the abortion group. The implications of this fact remain to be investigated.

Cytogenetics of human spermatozoa: correlations with sperm morphology and age of fertile men.

Sperm chromosomes from 15 fertile men were analyzed after fusion of their spermatozoa with zona-free hamster eggs. The total proportion of abnormal metaphases as well as the proportions of aneuploidy and structural aberrations were calculated for every man and examined for linear correlations with [1] sperm morphology and [2] the age of the persons studied. A positive correlation between the cytogenetic parameters and the percentage of abnormal sperm morphology was not evident, suggesting that assessment of sperm morphology cannot be used as an indicator of chromosomal damage in human spermatozoa. In contrast, there was a more distinct positive correlation between the age of donors and the three cytogenetic parameters studied.

Is there any correlation between follicular fluid hormone concentrations, fertilizability, and cytogenetic analysis of human oocytes recovered for in vitro fertilization?

Constituents of FF have been suspected to influence fertilizability and cytogenetic constitution of human oocytes. We therefore analyzed the FF concentrations of E2, P, T, and PRL for 114 oocytes recovered for IVF. Forty-six of these oocytes were fertilized and transferred to the maternal uterus. Among the unfertilized gametes, 27 were not analyzable, 30 were normal haploid, and 11 were classified as abnormal. There was no significant difference between fertilized and unfertilized oocytes for FF concentrations of E2, P, T, and PRL and for the E2:P ratios. Similarly, we detected no significant difference between normal and abnormal oocytes for these parameters.

First pregnancies after intravaginal transport and partial zona dissection of human oocytes.

We report on 72 couples with previous failure of fertilization during several trials of conventional IVF. For the present study, a part of the aspirated oocytes was subjected to partial zona dissection before insemination, thereby achieving 7 pregnancies during 104 cycles. Normal in vitro insemination of the remaining zona-intact oocytes was unsuccessful again. Because microinsemination techniques, in contrast to routine IVF, are not yet offered by many clinics, we developed the following new concept: hormonal stimulation, sonographic control of follicular growth, aspiration of oocytes, and sperm preparation were performed at the usual place (Munich), whereas partial zona dissection and control IVF were accomplished in another laboratory (Ulm). To transport the gametes between these two institutes, we modified the previously reported intravaginal culture of inseminated oocytes by transferring oocytes and spermatozoa into separate plastic capsules. In our view, this procedure offers an opportunity for infertile couples living too far away from a microinsemination center to afford long daily journeys or even several overnight stays just for superovulation treatment. The fertilization and PRs in our study are comparable with those reported in the literature.

Tetraploidy and partial endoreduplication in a tripronuclear zygote obtained after intracytoplasmic sperm injection.

OBJECTIVE: To describe a peculiar combination of cytogenetic abnormalities in a tripronuclear zygote obtained after intracytoplasmic sperm injection (ICSI). DESIGN: Case report. SETTING: A university hospital. PATIENT(S): A couple with a 4-year history of primary infertility. Intracytoplasmic sperm injection was performed because of male factor infertility (oligoteratozoospermia). INTERVENTION(S): Ultrasound-guided transvaginal follicular aspiration. MAIN OUTCOME MEASURE(S): Chromosomal karyotype of a tripronuclear one-cell zygote. RESULT(S): Unexpectedly, a tetraploid [92,XXYY, end3, -18, end18] chromosome complement was found, indicating injection of a diploid spermatozoon carrying two Y chromosomes. The parental origin of the other abnormalities could not be determined. The missing chromosomes may be attributed either to a hypodiploid [44,YY,-18,-18] sperm cell or to a hypohaploid [22,X,-18] oocyte. The exact tetraploid count was restored by endoreduplication of two chromosomes. This event could have occurred in one and the same or in two different pronuclei. CONCLUSION(S): Cytogenetic analysis of multipronuclear zygotes appears useful for assessing the incidence of chromosomal abnormalities at the earliest stage of conception. In addition to other methods, it also may contribute to evaluation of the transmission of aberrations by spermatozoa from infertile men.

Doppler sonographic findings and their correlation with implantation in an in vitro fertilization program.

In 45 women from an in vitro fertilization (IVF) program, the uterine and ovarian blood flows were investigated by vaginal Doppler sonography. The resistance index was used to evaluate the blood pattern. When comparing the patients who became pregnant after embryo transfer (ET [group I, n = 12]) with those who did not conceive (group II, n = 33), it is evident that in group I the vascular resistance of the uterine arteries is significantly lower on the day of follicular aspiration. No differences could be detected in the ovarian vessels. The data obtained so far suggest that the receptivity of the endometrium is a crucial factor for successful implantation. In the final analysis, this can be appraised not only on the basis of morphological but also of hemodynamic parameters.

Influence of smoking on fertility in women attending an in vitro fertilization program.

OBJECTIVE: To investigate the influence of cigarette smoking of women on the fertilization and pregnancy rates obtained by IVF treatment. PATIENTS: One hundred ninety-seven infertile, otherwise healthy women who entered an IVF program for the first time. SETTING: Fertility unit at the Women's University hospital of the University of Ulm, Ulm, Germany. INTERVENTIONS: The study population consisted of 197 women (23 to 39 years old) who were divided into the following groups: nonsmokers (n = 68), passive smokers (n = 26), and active smokers (n = 103) according to the cotinine concentration measured in follicular fluid. The reason for infertility was strictly a tubal factor with apparently normal ovulatory cycles. To guarantee an objective recording of tobacco smoke exposure, the smoking habit was not determined by questionnaires, but by cotinine, the principal metabolite of nicotine. RESULTS: There were no significant differences in fertilization and pregnancy rates between the different groups. The E2 serum levels were decreased significantly in women who smoked when compared with the results obtained from nonsmokers and passive smokers. Overall, a strong negative correlation of the cotinine and E2 levels was observed (r = -0.65). CONCLUSION: The results suggest that there is no clinically detectable impairment of fertilization potential due to female smoking and that there is a greater influence on the outcome of IVF by other factors.

Lack of correlation between ultrasonography and histologic staging of the endometrium in in vitro fertilization (IVF) patients.

In 53 patients of an in vitro fertilization (IVF) program with unsuccessful fertilization of oocytes, an endometrial biopsy was carried out on the day of the intended embryo transfer. The results were compared with the thickness (assessed on the very same day by means of ultrasonography) and the echo pattern of the endometrium, which was classified into four grades (A to D). We found grade A in 16 cases (30%), grade B in 22 cases (41.5%) and grade C in 15 cases (28.5%); no endometrium was assessed as grade D. The distribution of histologic findings was not significantly skewed within the respective grading categories (only 37.5% of grade A endometria, 63.5% of grade B endometria and 66.5% of grade C endometria were in phase with the menses). The in-phase and out-of-phase endometria did not display significant differences in endometrial thickness (8.8 +/- 0.29 mm vs. 9.13 +/- 0.4mm). Neither the sonographically measured endometrial thickness nor the echo pattern correlated with the histologic findings, suggesting that ultrasonography is inadequate for drawing reliable conclusions about endometrial receptivity in an IVF program.

Serum specific protein 1 and beta-human chorionic gonadotropin concentrations in patients with suspected ectopic pregnancies.

To determine the predictive value of serum specific protein 1 (SP1) and beta-human chorionic gonadotropin (beta-hCG) in the diagnosis of ectopic pregnancy, blood was sampled from 82 patients referred for suspected early extrauterine pregnancies. Serum concentrations of SP1 and beta-hCG were determined by EIA and RIA. All patients with early pregnancies had detectable SP1 and beta-hCG levels. An intrauterine pregnancy was found in six patients, and an ectopic pregnancy was confirmed by surgical explorations in 32 of these women. The remaining 40 women had other non-pregnancy related complaints. In all pregnant patients, the beta-hCG RIA and SP EIA determinations were also found positive. This study demonstrates the high predictive value of serum SP1 and beta-hCG determinations with an acceptable sensitivity and specificity in the diagnosis of suspected early ectopic pregnancies. In addition, the practicability and the easy performance of the enzymoimmunometric SP1 determination renders this test superior over the radioimmunoassay for beta-hCG determination.

[Effect of different follicle puncture methods on hormone homeostasis in the woman. Studies within the scope of an in vitro fertilization-embryo transfer program]. / Der Einfluss unterschiedlicher Follikelpunktionsmethoden auf den Hormonhaushalt der Frau. Untersuchungen im Rahmen eines In-vitro-Fertilisation-Embryotransfer-Programmes.

Different methods for follicular puncture which were used in an in vitro fertilization-embryo transfer program have been analyzed in order to determine their influence on plasma levels of estradiol, progesterone, prolactin and beta-endorphins. Thirty-eight patients awaiting oocyte aspiration were randomized into a laparoscopic (n = 20) and an ultrasound-guided oocyte aspiration group (n = 18). No differences were observed as far as estradiol and progesterone levels and embryo transfer rates were concerned. When using the laparoscopic technique, prolactin levels were found to be elevated 1.5 times in comparison with the ultrasound-guided aspiration procedure, which is highly significant (p < 0.001). Also, beta-endorphins peaked at a significantly higher level during laparoscopy (27.8 vs. 20.7 mol/l, p < 0.001). The enhanced prolactin and beta-endorphin levels reflect an increased stress response during laparoscopy. With respect to less trauma and shorter duration of the procedure, we now perform the sonographically guided transvaginal aspiration technique in our program almost exclusively.

Conjoined human oocytes observed during assisted reproduction: description of three cases and review of the literature.

Conjoined oocytes derived from binovular follicles have been suspected to play a role in producing dizygotic twins, mosaicism, tetraploidy or chimeras. This issue is discussed by presenting three examples for conjoined oocytes observed in our programme of human assisted reproduction and by including a review of corresponding cases. Our material comprises two associated immature oocytes and two cases in which an immature oocyte was attached to a mature oocyte. One of the mature oocytes was fertilized and transferred after cleavage without resulting in pregnancy. In the literature, another fifteen descriptions of conjoined oocytes were found. Here, simultaneous fertilization of both gametes occurred only once and there was no evidence for pregnancies arising from binovular follicles. These data suggest that binovular follicles do not cause dizygotic twins or genetic abnormalities.

The contradictory information on the distribution of non-disjunction and pre-division in female gametes.

Valuable information on the cytogenetic constitution of female gametes has been deduced from the direct, so-called conventional analysis of oocytes remaining unfertilized in programmes of assisted reproduction. Additional, indirect conclusions have become possible by PGD of the polar bodies. Both techniques provided evidence for the co-existence of two aneuploidy-causing mechanisms during first maternal meiosis; non-disjunction (ND) of bivalents results in the loss or gain of whole chromosomes in metaphase II complements, whereas a precocious division (pre-division, PD) of univalents leads to the loss or gain of single chromatids. As to the distribution of ND and PD, however, direct oocyte chromosome studies and PGD tell surprisingly different stories. Moreover, first and second polar body analyses contradict the data derived from DNA polymorphism studies concerning the distribution of first and second meiotic division errors. An increased awareness of these problems appears necessary because important decisions are made on the basis of PGD results.

Frequency and patterns of premature sperm chromosome condensation in oocytes failing to fertilize after intracytoplasmic sperm injection.

PURPOSE: The purpose of this study was to evaluate reasons for fertilization failure after intracytoplasmic sperm injection as a part of internal quality control and to review corresponding previous data. METHODS: One hundred injected but unfertilized oocytes were fixed and examined after Giemsa staining. RESULTS: Three oocytes (3.0%) did not show the presence of a spermatozoon and two (2.0%) contained pronuclear structures. An intact spermatozoon was found in 25 cases (25.0%), whereas the sperm nucleus had undergone premature chromosome condensation (PCC) in 70 cells (70.0%). A modified classification system was established to characterize the different PCC patterns. CONCLUSIONS: PCC indicates a correct intracytoplasmic injection and excludes technical problems as a major reason for fertilization failure in the present study. A lack of oocyte activation due to cytoplasmic immaturity is considered responsible for the occurrence of PCC. A review of the literature shows that the role of sperm chromatin abnormalities in the process of nuclear decondensation needs further investigation.

Cytogenetics of human spermatozoa: what about the reproductive relevance of structural chromosome aberrations?

PURPOSE: The purpose was to review and appreciate data on structural sperm chromosome abnormalities obtained after fusion of human spermatozoa with zona-free hamster eggs. RESULTS: Breaks and fragments are the predominant sperm chromosome aberrations. In contrast to stable alterations, e.g., translocations, inversions, and deletions that will be transmitted unchanged into following cell generations, breaks, fragments, and some rearrangements have a reduced stability. In proliferating cell systems they will soon be eliminated through formation of lagging chromatin and micronuclei. Their relevance lies in a loss of genetic material or disturbance of cell division that may cause cell death. It is reasonable to assume a responsibility of such aberrations for early unrecognized conception loss. However, this interpretation is subject to criticism because an artifactual origin of sperm chromosome breakage cannot be ruled out. CONCLUSIONS: The incidence and relevance of structural sperm chromosome abnormalities will remain at issue unless additional and complementary information is provided. For this purpose, new strategies must be developed because further studies employing hamster eggs according to existing protocols will not help resolve the dilemma.

Cytogenetics of human spermatozoa. The frequency of various chromosome aberrations and their relationship to clinical and biological parameters.

Human sperm chromosomes can be visualized after their fusion with zona-free hamster eggs. We analyzed a total of 867 metaphases from 33 men that had been classified into three different groups. We present a detailed summary of the kind and frequency of chromosome aberrations we encountered. A comparison of our results with previous studies demonstrates considerable variations in numerical and structural chromosome anomalies in spermatozoa from normal, healthy men. The possible reasons for this are discussed. We reviewed some factors which are supposed to correlate with the occurrence of sperm chromosome aberrations; the effect of paternal age is still uncertain.

Maturation of a binuclear oocyte from the germinal vesicle stage to metaphase II: formation of two polar bodies and two haploid chromosome sets.

We report on a binuclear human oocyte that underwent maturation in vitro from germinal vesicle stage to metaphase II. Following extrusion of two polar bodies, the oocyte was processed for cytogenetic analysis which revealed two separate haploid chromosome sets accompanied by the corresponding polar body chromatin. Tentatively established karyotypes were 23,X and 23,X,ace, respectively. This condition could have resulted in a tripronuclear digynic zygote after monospermic fertilization.

[Cytogenetic analysis of sperm chromosomes in couples with habitual abortion]. / Zytogenetische Analyse der Spermienchromosomen bei Paaren mit habituellen Aborten.

Genetic factors, especially numerical chromosome anomalies, play an important role in embryonic loss. Since somatic cell analysis cannot assess the risk of errors arising de novo during germ cell maturation, we investigated, whether the male gametes from couples with habitual abortion (group HA) carry a higher rate of anomalies, than those from donors without reproductive dysfunction (group K). Chromosomes were demonstrated after fusion of sperm with zona-free golden hamster ova. Our results indicate no significant difference between the two groups for the total rates of aneuploidy (HA: 3.6%, K: 2.0%) and structural anomalies (HA: 15.3%, K: 7.0%). However, the levels of chromosome breaks and acentric fragments were significantly higher in the abortion group (5.8% and 8.1%, respectively, vs. 2.4% each in group K). The implications of this fact remain to be investigated.

[Cytogenetics of unfertilized or uncleaved oocytes within the scope of in vitro fertilization. Relations to hormone content of follicular fluid]. / Zytogenetik unbefruchteter oder ungeteilt gebliebener Eizellen im Rahmen der In-vitro-Fertilisation. Beziehungen zum Hormongehalt der Follikelflüssigkeit.

Constituents of the follicular fluid (FF) have been suspected to influence fertilizability and cytogenetic constitution of human oocytes. Therefore, we analysed the FF concentrations of 17 beta-estradiol (E2), progesterone (P), testosterone (T), and prolactin (PRL) of 114 oocytes recovered for in vitro fertilisation (IVF). 46 of these oocytes were fertilised and transferred to the maternal uterus. Among the unfertilized gametes, 27 were not analysable. 30 were normal haploid, and 11 were classified as abnormal. There was no significant difference between fertilised and unfertilized oocytes for FF concentrations of E2, P, T, and PRL and for the E2/P ratios. Similarly, we detected no significant difference between normal and abnormal oocytes for these parameters.