Is human chorionic gonadotropin directly involved in the regulation of human implantation?

The regulation of human implantation is not fully understood. hCG as one of the earliest embryonal signals may be a major regulator in the parakrine embryo-endometrial communication. The expression of full-length hCG/LH-receptor mRNA could be demonstrated in human endometrium throughout the follicular and secretory phase of the menstrual cycle. In contrast, in early pregnancy decidua only truncated variants could be detected. To investigate direct effects of hCG on the human endometrium, an intrauterine microdialysis device was developed to measure parakrine mediators within the uterine cavity in vivo. Using this system, hCG was applied in the secretory phase and the endometrial response was evaluated. The administration of hCG (500 IU/ml) provoked a significant inhibition of intrauterine IGFBP-1 and M-CSF, while LIF, VEGF and MMP-9 were significantly stimulated. Taken together there appear to be multiple direct effects of hCG on the endometrium that precede the classical endocrine role of the hormone.

VEGF plasma pattern in ovulation induction: evidence for an episodic secretion and lack of immediate effect of hCG.

Vascular endothelial growth factor (VEGF) is a potent stimulator of vascular proliferation and permeability. Ovarian granulosa cells have been identified as a major source of the cytokine and r-hCG was able to stimulate VEGF mRNA expression in vitro. In this study we have investigated the immediate effect of ovulation induction with hCG on peripheral VEGF levels in 6 women with primary infertility enrolled in the IVF/ET program. The patients underwent a 24-hour continuous blood withdrawal with sampling intervals of 15 minutes starting from 5 hours before ovulation induction with 10.000 IU hCG. Ovulation induction with hCG had no significant immediate effect on mean peripheral VEGF levels. However, VEGF plasma levels did exhibit significant episodic fluctuations with rapid increases every 90-120 minutes without any relation to circulating hCG levels. Taken together, the results of this study suggest that VEGF is released episodically and that systemic VEGF levels are not acutely altered by ovulation induction with hCG.

Peripheral levels of vascular endothelial growth factor (VEGF) are higher in gonadotropin stimulated as compared to natural ovarian cycles.

The purpose of the present study was to investigate the stability of vascular endothelial growth factor (VEGF) in plasma samples and the influence of ovarian hyperstimulation on systemic levels of VEGF. Stability assays for VEGF in plasma samples revealed significant increases following even short incubations of samples at room temperature (< or = 2 h, p < 0.001). To investigate a possible impact of controlled ovarian hyperstimulation (COH) on peripheral VEGF levels, serial blood collection over one menstrual cycle was performed in unstimulated as well as in gonadotropin-stimulated cycles for in vitro fertilisation/embryo transfer (IVF/ET) (10 women each). Peripheral levels for VEGF were significantly higher in gonadotropin stimulated cycles as compared to non-stimulated cycles (p < 0.001). There was no significant difference between follicular phase and luteal phase levels in either group. VEGF levels tended to correlate with the number of follicles detected by vaginal sonography prior to oocyte aspiration (p = 0.051). In conclusion, VEGF levels are elevated in gonadotropin-stimulated IVF/ET cycles as compared to natural cycles.

Novel insights into human endometrial paracrinology and embryo-maternal communication by intrauterine microdialysis.

The regulation of human implantation is still unknown. Evidence from mice suggests an essential role for several paracrine mediators but species differences with implantation in the human preclude the extrapolation of these concepts to humans. An intrauterine microdialysis device (IUMD), consisting of microdialysis tubing glued into a balloon catheter on one side and into a polypropylene tube on the other, allows a dynamic and accurate in-vivo measurement of uterine paracrine interactions in humans. Inserted into the uterine cavity in the form of a loop, it can be continuously perfused with saline to reveal a number of relevant cytokines and growth factors in uterine effluents of non-pregnant women in both follicular and luteal phases. These included interleukin (IL)-1alpha, IL-1beta, IL-6, leukaemia inhibitory factor (LIF), macrophage colony-stimulating factor (M-CSF), epidermal growth factor, vascular endothelial growth factor (VEGF), insulin-like growth factor binding protein-1 (IGFBP-1), prolactin, and human chorionic gonadotrophin (HCG). The source of intrauterine HCG is unclear since endometrial mRNA for the HCG beta-subunit is not revealed using reverse transcriptase polymerase chain reaction analysis. Applying urinary HCG locally via the IUMD profoundly alters endometrial secretory parameters. Prolactin, IGFBP-1, and M-CSF are significantly inhibited and VEGF is regulated in a biphasic manner involving early stimulation followed by inhibition of intrauterine levels. Use of the IUMD has thus shown that the urinary HCG preparations routinely used for ovulation induction and luteal support may directly alter endometrial function.

Substitution with testosterone as aromatizable substrate for induction of follicular maturation, estradiol production and ovulation in a patient with 17 alpha-hydroxylase deficiency.

17 alpha-Hydroxylase Deficiency (17 alpha-OHDS) is a rare defect of steroid biosynthesis, characterized by the inability to synthesize cortisol, androgens or estrogens, by the complete absence of follicular maturation, hypergonadotropic hypogonadism, primary amenorrhea and hypertension. Since the ovaries of such patients contain numerous primordial follicles, we hypothesized that the absence of spontaneous follicular maturation could be due to a lack of aromatizable substrate. To provide this substrate, testosterone was administered either by intra-ovarian injection or by vaginal administration. Ovarian stimulation was performed with human urinary gonadotropins. Follicular maturation and ovulation could be induced by this treatment, as determined from ultrasonography, the analysis of LH, estradiol and progesterone serum levels and the aspiration of oocytes from the mature follicles. Fertilization of these oocytes in vitro, however, did not succeed. We conclude that follicular maturation can be induced in 17 alpha-OHDS by gonadotropins when testosterone is provided as an aromatizable substrate and that estrogens are a necessary component of follicular maturation.

[Ovarian stimulation with gonadotropins in undetected tubal pregnancy within the scope of in vitro fertilization]. / Ovarielle Stimulation mit Gonadotropinen bei unentdeckter Eileiterschwangerschaft im Rahmen der In-vitro-Fertilisation.

The authors report on a patient who was pretreated with a depot GnRH-analog previous to exogenous gonadotropin stimulation before in-vitro fertilisation (IVF). The patient had an undiagnosed ectopic pregnancy. Folliculogenesis and the hormone profile were normal. In patients with patent tubes, contraception (non-hormonal) in the cycle previous to IVF should be performed.

[Extension of the concept of extracorporeal fertilization by cryopreservation of impregnated oocytes]. / Die Erweiterung des Konzepts der extrakorporalen Befruchtung durch die Kryokonservierung imprägnierter Eizellen.

Cryopreservation of human embryos within in-vitro fertilization treatment was already proposed in 1977 by Edwards and Steptoe. Today, this procedure is used worldwide by several teams as a standard method to enhance the success rate of IVF. In spite of this, there are many disadvantages of the freezing of embryos, furthermore it is legally prohibited in Germany. We report on a new concept for treatment, which includes the freezing of impregnated oocytes after IVF and their transfer in subsequent cycles. From 109 out of 120 patients who were treated with a long acting GnRH analogue or a contraceptive pill before IVF stimulation, "supernumerary" impregnated oocytes could be frozen. Twenty-three pregnancies resulted after immediate embryo transfer, and thirty patients, who returned for the transfer of cryopreserved oocytes, became pregnant. Neither the IVF pre-treatment, nor the stimulation in the transfer cycles of frozen oocytes with clomiphene citrate, influenced the success rate significantly. Combining the 53 pregnancies, a cumulative pregnancy rate of 49% can be calculated per IVF stimulation treatment. This means a doubling of the success rate, compared with routine IVF treatment. Although there are still problems to be solved, as, for instance, the insufficient implantation rate of 7% after the transfer of frozen/thawed cells, we consider cryopreservation of pronuclear oocytes a useful and promising supplement of IVF treatment. There is no further need for the freezing of embryos.

Internal quality control of semen analysis.

OBJECTIVE: To test a scheme for quality control of semen analysis. DESIGN: The reproducibility of assessment of sperm concentration, motility, and morphology was obtained for the same sample measured by different technicians (between or intertechnician variation) and for different samples assessed by each technician with time (within or intratechnician variation). SETTING: Andrology Laboratory. PATIENTS: Semen samples were analyzed from all patients attending the clinic. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Within technician and between technician coefficients of variation for concentration, motility, and morphology. RESULTS: When 100 sperm were routinely assessed, both intratechnician variation, as assessed from the precision of duplicate measurements, and intertechnician variation revealed hyperbolic curves with increasing variation at low percentages (less than 20) of motile or morphological forms. When these low values were excluded, mean intratechnician variations were 5.0%, 8.4%, and 2.8% for concentration, motility, and morphology, respectively, and mean intertechnician variations were, respectively, 6.1%, 5.6%, and 5.6%. Similar mean intertechnician variation for morphology was obtained for repeated assessment of prestained (7.3%) or presmeared (5.9%) slides. The use of cryopreserved semen to monitor longitudinal changes in the technicians' assessments revealed variations of 8.1% to 12% in concentration and 9.7% to 14% in motility. Computing the monthly means for sperm concentration, motility, and morphology over a 4.5-year period revealed a marked reduction in percentage of normal morphological forms, unrelated to the sperm count or mean age of the men attending the clinic. This was shown to be a result of a shift in the assessment by technicians. CONCLUSIONS: Quality control is necessary and possible in an andrology clinic.

Hyaluronic acid as a medium for human sperm migration tests.

Hyaluronic acid was used as a substitute for human cervical mucus as a medium for sperm migration since its viscosity, molecular weight and structure are similar to constituent glycoproteins of human cervical mucus. Parameters of sperm motility were comparable in human cervical mucus and hyaluronate as measured by a computerized sperm motion analysis system (Hamilton-Thorn 2030 and Strömberg-Mika System). Sperm migration rates were also similar in human cervical mucus, bovine mucus and hyaluronic acid and the reproducibility was best in hyaluronic acid. Sperm survival for 24 h was maintained in human cervical mucus and hyaluronic acid but not in bovine mucus. The use of hyaluronic acid in sperm migration tests is recommended.

'9 + 0' axoneme in spermatozoa and some nasal cilia of a patient with totally immotile spermatozoa associated with thickened sheath and short midpiece.

A patient is described with 100% immotile spermatozoa. The sperm cells lack the central pair of microtubules or the complete axoneme. This defect is associated with a thickened fibrous sheath and a shortened midpiece containing defective mitochondria. Fifteen per cent of the nasal cilia of this patient lack the central pair of microtubules. Eighteen similar cases have been described in the literature suggesting that this association of ultrastructural defects may be considered a syndrome.

External quality control in the andrology laboratory: an experimental multicenter trial.

An external quality control study for semen analysis was performed involving 10 andrology laboratories in geographically separate locations. For the evaluation of sperm concentration, eight samples with different concentrations, and for the assessment of sperm morphology three slides prepared from different semen samples were distributed. Sperm motility was evaluated in five samples delivered cryopreserved to the participants. The coefficients of variation (CVs) for sperm counts varied with the sperm concentrations showing highest variability for low and lowest for high concentrations (range 23% to 73%). The CV for sperm morphology ranged from 25% for normal heads to 87% for abnormal midpieces. The CV for motility of sperm was 21%. For comparison the mean CVs for internal quality control were as follows: 10% for concentration, 8% for morphology (normal heads), and 8% for motility.

Computerized semen analysis with sperm tail detection.

We evaluated a recently developed computerized semen analyser that detects spermatozoa not only by the criteria of size, contrast and movement but also by the morphological characteristics of the sperm tail. Comparison of the sperm concentration in 33 semen samples measured by conventional and by computerized semen analysis, as well as by flow cytometry, showed acceptable agreement between all three methods, although the mean differences and standard deviations were less for conventional than for computerized analysis when compared to flow cytometry as a reference method. Motility estimates were lower by the computer system for values between 1 and 40%. Higher motilities showed no systematic error. In conclusion, the improved algorithms for sperm detection yield more reliable data for sperm concentration and motility than previous systems of computerized semen analysis.

The influence of relaxin on motility of human sperm in vitro.

Sperm of healthy men were incubated in an IVF medium with relaxin at concentrations of 3, 30, 300 and 3000 ng ml-1. Immediately after addition of relaxin and 60 and 120 min later motility, progressive motility, mean path velocity, mean progressive velocity, mean linearity and mean lateral head displacement were measured with the Hamilton-Thorn motility analyser. Neither immediately after relaxin addition, nor after 60 or 120 min, was an improvement of sperm motility observed at any concentration.

Evaluation of the Hamilton-Thorn 2030 motility analyser for routine semen analysis in an infertility clinic.

In routine semen analysis of 242 patients the values of sperm concentration, sperm motility and progressive sperm motility were measured with the Hamilton-Thorn semen analyser (HT) and compared to the data obtained by conventional semen analysis according to the guidelines of the WHO. Overall, the HT gave higher values for sperm concentration (mean difference 21.7 +/- 46.2 x 10(6) ml-1, mean +/- SD). Motility values showed a correlation of 0.67 (slope 0.94, P less than 0.001) but were estimated lower by the HT than by conventional analysis (mean difference 7.3 +/- 21.7%); this was caused by the overestimation of sperm concentration. In the range from 0 to 50% motility the HT yielded lower values and higher values from 50 to 100% motility. Progressive motility values of the HT agreed better with conventional analysis (WHO categories a + b): the mean difference of all values was 0.5 +/- 19.5% (r = 0.74, slope = 1.0). The mean lateral head displacement measured by the HT increased with increasing sperm path velocity, but other significant correlations between sperm movement parameters could not be demonstrated. In many instances round cells and debris could not be distinguished from normal sperm. In conclusion, the HT system cannot replace conventional semen analysis in routine diagnosis.

Evaluation of the semi-automated Autosperm semen analysis system. I. Accuracy and comparison with the conventional method and the automated Hamilton-Thorn system.

The accuracy of measurements by the semi-automated Autosperm (Amsaten N.V.S.A. Corp., DePinte, Belgium) semen analysis system was assessed by recounting and manually tracking sperm recorded on videotape during analysis of 51 ejaculates. Mean inaccuracies in the analysis of sperm concentration and percentage motility were 15% and 22%, respectively. Measurements of sperm movement characteristics relied on the skill of the operator and discrepancies (means around 10%, maximum 57% to 184%) depended on the straightness of sperm paths. Although less expensive than the fully automated system, semen analysis by Autosperm is a subjective and labor-intensive method. Furthermore in comparison, data obtained using Autosperm also provide less information, and agreements of matched data with those obtained by the conventional methods were not significantly better.

Bias to routine semen analysis by uncontrolled changes in laboratory environment--detection by long-term sampling of monthly means for quality control.

To detect systematic bias in the results of routine semen analysis over time, monthly means of semen parameters determined by the recommended WHO methods were computed. The analysis was based on a total sample size of 1784 ejaculates and included 18 months of observation. In addition to slight changes of morphology estimates caused by a change of laboratory staff, a major bias in the measurement of sperm motility could be detected. This observation triggered a search for changes in protocols not previously given the required attention. It revealed that the newly introduced use of polypropylene syringes with a mounted needle for accurate measurement of seminal volume impaired sperm motility. More detailed investigation by computerized sperm motion analysis in 10 semen samples treated simultaneously in different ways revealed that predominantly it was the needle which caused the drop in proportion of motile sperm (glass cylinder: 50.3 +/- 4.1% vs. syringe + needle: 26.6 +/- 5.3%; mean +/- SEM) and not the contact with the plastic material alone (syringe alone: 43.4 +/- 4.8%). Other motion parameters such as curvilinear velocity (36.0 +/- 1.6 microns/sec), linearity (78.5 +/- 8.4%) and lateral head displacement (3.8 +/- 0.9) were not influenced by the different methods of handling. The results indicate that long-term sampling of monthly means may serve as part of a quality control scheme in semen analysis.