Spermoculture: improvement of the bacteriological quality of samples by direct verbal counseling before semen collection.

OBJECTIVE: To compare the bacteriologic quality of samples after direct verbal or written counseling for semen collection. DESIGN: Prospective randomized comparative study. SETTING: Laboratories of biology of reproduction and microbiology in university hospitals. PATIENTS: Male partners of infertile couples on their first attempt to collect semen were assigned randomly into two groups. INTERVENTION: In one group (n = 52) the patients were given direct verbal instruction by one of the investigators on how to avoid bacterial contamination during semen collection. In the other group (n = 52) the patients were asked to follow the usual written instructions. MAIN OUTCOME MEASURE: Bacterial count, number of species, classification of the samples in positive, negative, and noninformative cultures. RESULTS: Direct verbal teaching significantly reduced the bacterial count (2.06 +/- 1.6 versus 3.29 +/- 1.6 log cfu/mL; mean +/- SD) and number of species (1.44 +/- 2.02 versus 3.25 +/- 2.25), increased the percentage of sterile cultures (59.6% versus 23.1%), and decreased the percentage of noninformative cultures (29.9% versus 50%). CONCLUSION: Direct verbal counseling on how to avoid bacterial contamination during semen collection significantly improves the bacteriologic quality of the samples.

Motility and fertilizing capacity of epididymal human spermatozoa in normal and pathological cases.

OBJECTIVE: To investigate different factors involved in the outcome of in vitro fertilization (IVF) with epididymal spermatozoa in cases of vas deferens agenesis, with particular emphasis on sperm movement parameters. DESIGN: Prospective study in which sperm movement characteristics, level of puncture, and sperm preparation technique were studied relative to the outcome of IVF. SETTING: Département de Gynécologie, Oncologie Gynécologique, Sénologie, Médecine de la Reproduction, Hôpital Edouard-Herriot, Lyon France. PATIENTS: Thirteen couples underwent 15 IVF cycles. In 14 cycles (12 patients) epididymal spermatozoa could be recovered by surgical punctures. Epididymides were taken from 10 subjects in state of cerebral death to study epididymal spermatozoa under physiological status of the epididymis. RESULTS: The cleavage rate in IVF was low (25/158: 15.8%). In seven cases, embryo transfer was possible, leading to two twin pregnancies (4 infants born). Fertilization could be achieved with spermatozoa from the epididymis caput. Motility was paradoxically higher in the proximal epididymis than in the distal epididymis. In healthy epididymides, spermatozoa collected from the caput did not exhibit any progressive motility, whereas progressive motility was always present in the epididymis corpus. CONCLUSIONS: Our study showed that forward motility could be observed in the epididymis caput in patients with congenital absence of the vas deferens and that fertilization and ongoing pregnancies could be obtained from caput spermatozoa. On the other hand, in physiological status of the epididymis, forward motility was observed only as from the corpus area, confirming observations in other species.

[Semen evaluation: technic of study]. / Spermogramme: technique de réalisation.

Sampling precautions are particularly important in the study of sperm: the length of sexual abstinence, complete collection of the ejaculate, sampling by masturbation in the laboratory. The examination of sperm consists of different steps: measurement of the ejaculate volume, evaluation of the mobility, speed, and propulsion direction of the spermatozoa, study of spermatozoon concentration, estimation of the percentage of live spermatozoa, morphological examination of the spermatozoa, including an enumeration of the abnormal forms, evaluation of spermatozoon survival in vitro for 24 h by monitoring the percentage of mobility every 2 h. This examination can be complemented by biochemical and enzymatic assays of: fructose, zinc, carnitine, citric acid, acid phosphatase, alpha glucosidase, which serve as tracers of associated secretions.

[Separation of spermatozoa using Percoll gradients: value for in vitro fertilization]. / Séparation des spermatozoïdes en gradient de Percoll: intérêt pour la fécondation in vitro (FIV).

Male infertility can be successfully treated by In Vitro Fertilization (IVF) and Embryo Transfer. However in case of oligo asthenospermia it is often difficult to select enough spermatozoa with good motility for the oocytes insemination. To cope with this problem, in 27 IVF cycles, sperm selection was performed by centrifugation on discontinuous Percoll density gradient. This method was demonstrated to be more efficient than swim-up migration to select motile sperm. The cleavage rate (63%) and the pregnancy rate per cycle (22%) were both satisfying. Centrifugation on discontinuous Percoll gradient was also found to be highly efficient for the removal of bacterial contaminants from semen (20 IVF cycles); the cleavage rate (66%) and the pregnancy rate per cycle (35%) were also quite satisfying. It can be concluded that centrifugation on discontinuous Percoll gradient is a useful method for sperm preparation in IVF in case of oligo-asthenospermia and in case of bacterial contamination in semen.

[Therapeutic hopes in excretory azoospermia and genetic risks in congenital aplasia of the vas deferens]. / Espoirs thérapeutiques dans les azoospermies excrétoires et risques génétiques dans l'asplasie congénitale des canaux déférents.

Therapeutical solutions now can be proposed to some excretory azoospermia, using in vitro-fertilization with epididymal sperm. The encouraging results obtained with this new approach should be analyzed with the genetic risk, sometimes encountered in the specific form of azoospermia due to the congenital absence of the vas deferens. This abnormality is to day supposed to represent a moderate form of cystic fibrosis (CF), corresponding to a genital phenotype of this disease. This suggestion has been firstly induced by similar anatomical findings in the male individuals presenting a classical form of CF. The hypothesis has mainly been confirmed by the real progress in the genetic analysis of this disease. So an abnormally high percentage of known mutations of CF has been demonstrated in the patients with congenital absence of vas deferens. Other arguments such as positive sweat chloride tests, high percentage of sinusitis or presence of anti-pseudomonas antibodies, reinforce this hypothesis. It is the reason why a clinical and biological check-up, prior to any decision of therapy for infertility, in this specific indication should be done in order to propose a genetic counselling to the couple.

[Seasonal variations of cellular composition of human semen (author's transl)]. / Variations saisonnières dans la composition cellulaire du sperme humain.

Sperm samples taken fortnightly from 14 young europeans over a period of 8 months show evidence of a seasonal variation in the concentration of spermatozoa and in the frequency of abnormal sperm (teratospermy). Both factors are highest in winter and lowest in springtime. A study of 3,000 spermograms established during consultations for sterility over the last 2 years did not clearly reveal the same trends, however, a statistical analysis of the results leads to the conclusion that several hundred examinations per month would be needed to show a significant seasonal variation in such a heterogeneous sample.

[Study of different components in spermatic function after failure of in vitro fertilization]. / Etude des différents composants de la fonction spermatique après échec de fécondation in vitro.

The fertilizing potential of sperm depends on numerous properties which influence its functional competence. In order to understand the causes of IVF failure, we studied some of these properties-movement characteristics, hyperactivated motility, acrosome reaction-for infertile sperm (0% in vitro fertilization) and compared to those of fertile sperm (> or = 50% fertilization, control group). Movement characteristics, assessed by video-micrographic analysis, are significantly different for the 2 groups: infertile sperm register the lowest values for motility characteristics and hyperactivation. The addition of ionophore A23187 to the medium improves the hyperactivation and the acrosome reaction, the values for the latter, however, remain significantly lower compared to the control. These results are in favor of a detailed study of motility and induced acrosome reaction as complement for the diagnosis of male infertility.

In vitro behavior of human nonfertile semen in isoosmotic and hyperosmotic media.

High-ionic-strength media are known to favor rapid capacitation and acrosome reaction of spermatozoa in vitro. The influence of isoosmolar (N-BWW) and hyperosmolar (H-BWW) media on the percent motility, forward progression, and zona-free hamster egg penetration has been investigated for 148 semen from patients consulting for male or idiopathic sterility. After 15 h of incubation, the hyperosmotic medium had no significant detrimental effect on the percent motility of spermatozoa, although the forward progression was better maintained in the isoosmolar medium. In general egg penetration was increased when spermatozoa were treated with the hypertonic medium, and in particular 5 patients whose semen showed no penetration when treated with N-BWW scored greater than 12% when treated with H-BWW. For the 23 subjects who had an IVF at the same period, a good agreement with zona-free egg penetration was found in 70% of the cases.

[Therapeutic use of LH-RH analogs]. / Utilisation thérapeutique des analogues de la GnRH.

The development of GnRH analogues enabling a hypophyseal desensitization with suppression of the gonadotrophic activity has opened new therapeutic possibilities in early puberty of central origin, metastatic prostate and breast cancers, endometriosis, fibromyomas and the syndrome of polycystic ovaries. This retrospective study concerning 93 cycles of in vitro fertilization between March and June 1987, compares the results of the combination of a GnRH agonist (busereline 0.3 mg s.c. x 2 days) with HMGs according to a "long protocol" or a "short protocol". For a mean stimulation time by HMG equivalent (13 days), the number of HMG vials is higher in case of a long protocol (40 vs 30). The mean estradiol level, on the morning of the induction is identical (2,096 pg/ml), as well as the mean number of ovocytes collected (6/tap). The segmentation rate appears to be better in case of long protocol (70% vs 45% in female indications), but there is no significant difference between the pregnancy rates (20 to 30%). The short protocol, less costly and simpler, seems therefore interesting in FIV, with patients without SOPK.

Improvement of survival and fertilizing capacity of human spermatozoa in an IVF programme by selection on discontinuous Percoll gradients.

Two techniques for the separation of spermatozoa were compared: swim-up migration (SUM) and centrifugation on a discontinuous Percoll gradient (CPG). Their respective effects on sperm motility were analysed by computer-assisted videomicrography in either normal or asthenozoospermic groups. In both groups, there was no difference in any of the motion parameters between the two treatments after 1-h incubation. However, a clear difference was observed after 24 h when excellent motility was retained only in the CPG-treated group. A total of 350 ejaculates were produced by the husbands of women undergoing oocyte retrieval in an IVF programme. Spermatozoa were treated by CPG when the infertility was due to poor quality spermatozoa (n = 91), when there was a known previous history of semen infection (n = 73) or when frozen semen, originating from a donor, was used (n = 36). In all other cases (n = 150), spermatozoa were treated by SUM. The cleavage rates obtained were 32.2, 70.1, 60.9 and 68.6% respectively in the four categories. The clinical pregnancy rates per oocyte retrieval were 19.8, 31.5, 22.2 and 18.0% respectively. Forty-eight births occurred in the CPG group: 28 boys and 20 girls, all normal. We conclude that CPG is useful, both in cases of poor semen quality and in tubal infertility, in which the clinical pregnancy rate increased significantly from 18.0 to 31.5%.

[beta2-Microglobulin in human seminal fluid (author's transl)]. / La beta2-microglobuline dans le liquide séminal.

The expression of human beta2-microglobulin (beta2m) on human spermatozoa cell surface was investigated by cytotoxicity and immunofluorescence tests. beta2m was also found in human seminal fluid by use of immunochemical techniques. Seminal fluids obtained from couples with infertility problems were tested for beta2m, albumin and total proteins concentration. A higher level of beta-2m was found in azoospermia compared with that of normal sperms.

Individual characteristics of human spermatozoa.

We have investigated the variability of some characteristics of a sperm sample in 18 young volunteers in good health who were examined twice per month over a period of two years, and in about one hundred men undergoing treatment for sterility and for whom more than 4 spermiograms had been established during the past three years. The results of statistical analysis indicate that the most characteristic individual feature is the concentration of spermatozoa.

[Value of plasma progesterone (rapid assay) during ovarian stimulation for in vitro fertilization using LHRH agonists and hMG]. / Intérêt de l'étude de la progestérone plasmatique (dosage rapide) au cours de la stimulation ovarienne pour fécondation in vitro utilisant les agonistes de LHRH et les hMG.

We have studied all the cycles (= 276) induced for in vitro fertilization using a LHRH agonist and the hMG (113 short protocols, 163 long protocols) from 01.90 to 07.90 to know the plasmatic profiles of the estradiol (E2), of the LH and specially of the progesterone (P) between J-7 and the day of the hCG (= J0). We observe in the short protocol an elevation (effect flare up) and afterwards a fall concomitant of the P and of the LH up to J-5, J-6 (effect of pituitary down regulation), in the short and long protocols a progressive elevation of J-3 up to J0 of the P parallel to E2, so that the LH remain stable or decrease. The LH of J0 is higher in short protocol than in long protocol (10.14 +/- 2.9 versus 4.72 +/- 1.3 Ul/l, p < 0.00001). We found the same progressive elevation of the P of J-3 up to J0 in pregnant and not pregnant patients. The surveillance of the P coupled to the E2 and to the follicular ultrasonography could help in the choice of the day of the hCG and avoid a post-maturity of oocyte. The consequences of the elevation of P In the morning of J0 have been study for P levels < 1 ng/ml (73 cases) and P > or = 1 ng/ml (203 cases) and afterwards for progressive elevation of P levels (1.5-2-2.5-3 ng/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

Morphological and cytogenetic observations of unfertilized human oocytes and abnormal embryos obtained after ovarian stimulation with pure follicle stimulating hormone following pituitary desensitization.

Morphological and cytological observations of 189 unfertilized oocytes and 40 abnormal embryos obtained from 32 patients in a routine in-vitro fertilization programme were performed. Both the oocytes and the embryos were mounted whole to preserve the original topology of all the structural elements. With the applied protocol of ovarian stimulation associating pituitary desensitization and follicle stimulating hormone stimulation, a high degree of immaturity of the unfertilized eggs was observed in comparison with previous reports. This immaturity was deduced from the higher incidence of unfertilized eggs arrested at the germinal vesicle or metaphase I stage, as well as metaphase II oocytes with multiple metaphase plates. Nine triploid and four tetraploid embryos were analysed: except for one tetraploid embryo, all the polyploid embryos cleaved. The percentages of mononucleated blastomeres in these polyploid embryos were 57 and 27% respectively. We also analysed 21 cleaving diploid embryos which exhibited a high degree of fragmentation. No more than 40% of the blastomeres contained a single nucleus. Moreover, in only one of the 21 diploid embryos could all the blastomeres be considered normal.

Relationship between semen characteristics, alpha-glucosidase and the capacity of spermatozoa to bind to the human zona pellucida.

Unfertilized oocytes from an in-vitro fertilization programme were stored in different saline solutions and then utilized in a zona binding assay (ZBA). The four saline solutions tested were identical with regard to the capacity of the zona to bind spermatozoa provided by healthy donors. Spermatozoa from 150 infertile patients were tested in the ZBA. The number of spermatozoa bound to the zona correlated positively with sperm concentration, the percentage sperm motility and the percentage of morphologically normal spermatozoa. The population was then divided into two groups according to the level of alpha-glucosidase activity, an epididymal marker. The average number of spermatozoa bound to the zona was diminished in the group with low alpha-glucosidase activity, even when considering strictly equivalent classes of sperm concentration, motility and morphology, respectively.

Transfers of frozen-thawed human embryos in cycles stimulated by HMG.

A total of 130 transfers of frozen-thawed (F-T) human embryos was carried out after moderate ovarian stimulation with human menopausal gonadotrophin (HMG). Embryos were replaced 3 days after the spontaneous luteinizing hormone (LH) surge or 4 days if ovulation was induced by human chorionic gonadotrophin (HCG). Embryos were thawed a few hours prior to transfer. One-hundred-and-twenty-three transfers were effective and 23 pregnancies were achieved. The rate of ongoing pregnancies per transfer was 17.9% (22/123). The survival rate of embryos originating from cycles stimulated by a combination of an LHRH analogue and HMG in a long protocol (LA-HMG protocol) was significantly lower when compared with the rate of embryos retrieved from clomiphene citrate-HMG (CC-HMG protocol) stimulated cycles (52 versus 67%, P less than 0.05). When fresh embryos originated from cycles stimulated with an LHRH analogue and HMG in a short protocol (SA-HMG protocol), the survival rate was not affected (59 versus 67%, NS). Although the difference was not significant, the ongoing pregnancy rate per transfer according to the three protocols from which the embryos originated seemed to be better with the SA-HMG protocol: 16% with the CC-HMG protocol, 14.5% with the LA-HMG protocol versus 27.6% with the SA-HMG protocol. The success rate was independent of the number of F-T transferred embryos if at least one embryo with 100% intact blastomeres was replaced.

[In vitro fertilization failures related to a sperm hyperactivation defect: indication for microinjection?]. / Echecs de FIV en relation avec un défaut d'hyperactivation des spermatozoïdes: indication de micro-injection?

Development of hyperactivated motility is considered as necessary for penetration through zona pellucida. In a study concerning 114 IVF attempts, we could not establish a significant correlation between fertilization rate and hyperactivation rate (HA). On the contrary, most fertilization failures (16/21) we found for HA values < 10%. 10 of such couples could benefit of ICSI for the next attempt: fertilization rates were strictly similar to the general rate observed for ICSI in our group (48%).