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Flow cytometric sorting of frozen-thawed spermatozoa in sheep and non-human primates.

Research was conducted in sheep to determine an effective preparation method for high-purity sorting of frozen-thawed spermatozoa. The efficacy of sorting frozen-thawed spermatozoa was then investigated in several non-human primate species. An aliquot of each ejaculate (three rams, three ejaculates per ram) was processed as a fresh control (FRESH). Frozen spermatozoa were thawed and prepared for sorting by no further processing (FT-NEAT), washing (FT-WASH) or gradient centrifugation (FT-GRADIENT) and evaluated for motility at 1 h post-staining and motility and acrosomal status at 0 and 4 h post-sorting. Samples were analysed using a high-speed cell sorter. High levels of purity for X- and Y-enriched samples were achieved for all treatments (85-92%). The percentage of motile spermatozoa before sorting was lower (P < 0.05) for frozen-thawed samples (FT-NEAT: 32.7 +/- 2.5%; FT-WASH: 32.2 +/- 3.3%; FT-GRADIENT: 73.9 +/- 3.7%) compared with FRESH (83.3 +/- 1.2%). Post-sorting, the percentage of motile spermatozoa before and after incubation for FT-NEAT (60.0 +/- 5.1% and 27.2 +/- 6.1% for 0 and 4 h, respectively) was lower than that for FRESH (87.8 +/- 0.9% and 83.3 +/- 1.2% for 0 and 4 h, respectively; P < 0.05), FT-WASH (80.0 +/- 2.4% and 71.7 +/- 3.6% for 0 and 4 h, respectively; P < 0.05) and FT-GRADIENT (84.4 +/- 1.3% and 77.2 +/- 1.7% for 0 and 4 h, respectively; P < 0.05). Vanguard sperm migration distance through artificial cervical mucus was lower (P < 0.05) for FT-NEAT (17.7 +/- 1.7 mm) compared with FT-WASH (29.1 +/- 3.8 mm) and FT-GRADIENT (28.4 +/- 2.0 mm) and similar (P < 0.05) to FRESH (23.7 +/- 1.8 mm). Sample preparation using a modified wash method enabled high-purity sorting (range 86-97% purity) of frozen-thawed epididymal spermatozoa in the baboon (Papio hamadryas), common marmoset (Callithrix jacchus) and common chimpanzee (Pan troglodytes). For all non-human primate species, sorted spermatozoa were progressively motile (marmoset: 20.5 +/- 5.5%; baboon: 37.5 +/- 2.5%; chimpanzee: 73.0 +/- 2.0%), acrosome intact (marmoset: 68.5 +/- 7.5%; baboon: 89.5 +/- 1.5%; chimpanzee: 84.0 +/- 1.0%) and able to penetrate an artificial cervical mucus. In summary, high-purity sorting of frozen-thawed ram and non-human primate spermatozoa with recovery of progressively motile, acrosome-intact spermatozoa was possible after processing to remove cryodiluent.

Animals↗

A randomized comparison of intrauterine and intraperitoneal insemination in the treatment of infertility.

OBJECTIVE: To compare the pregnancy rates (PRs) between intrauterine insemination (IUI) and intraperitoneal insemination (IPI) in women receiving comparable ovarian stimulation in couples who were infertile for various infertility conditions. METHOD: Fifty-four couples were treated. Of these, 25 were diagnosed as having unexplained infertility, 21 as having male infertility and 8 as having cervical mucus hostility. RESULT: Nine pregnancies occurred with 89 cycles of IUI and 6 pregnancies with 85 cycles of IPI, giving an overall PR of 28%. Twelve of the 15 pregnancies occurred during the first treatment cycle and three during the second treatment cycle. There was no significant difference in the (PRs) between the IUI and IPI (P > 0.25). CONCLUSION: Both procedures combined with superovulation proved to be simple, inexpensive safe and worthwhile for infertile couples with unexplained infertility, male infertility and cervical mucus hostility.

Adult↗

Progestin-releasing intrauterine systems.

The levonorgestrel-releasing intrauterine system (LNG IUS) is an effective method for contraception. It has a strong antiproliferative action on the endometrium. The endometrium is transformed under the influence of local levonorgestrel and becomes unresponsive to ovarian estrogens. This process is associated with progressive reduction of menstrual blood loss and menstrual duration. Scanty and irregular bleeding and/or spotting is usual during the first 3 to 4 months. The reduction of menstrual blood loss continues and after the first 9 months many women have no bleeding at all. However, they have normal ovarian function. The absence of bleeding is a result of the local antiproliferative action of the LNG IUS on the endometrium, which is also responsible for many health benefits during the use of this method. As with oral contraceptives, the risk of pelvic inflammatory disease is reduced, because of reduced menstrual blood loss, endometrial suppression, and thickening of the cervical mucus. There are some steroidal side effects: mood changes, oily skin, and acne. Weight increase is similar to that associated with copper intrauterine devices: 500 g per year over 5 years. Users should be told that the LNG IUS does not prevent sexually transmitted infection, and therefore women at risk should also use condoms for their protection.

Acne Vulgaris↗

Sperm processing: glass wool column filtration.

The cervical mucus selectively allows only progressively motile sperm of normal shape and size to penetrate and migrate through the cervix. The glass wool column filtration (GWCF) method for sperm processing, proposed in 1977 and later modified and standardized in 1986, selects such a sperm population in vitro. Sperm recovered from GWCF is significantly higher in quality than the original ejaculate. GWCF processing is reliable and highly repeatable, and when compared to other sperm processing techniques, GWCF yields significantly higher sperm quality. Sperm penetration into denuded hamster oocytes improves significantly with GWCF, while binding to human zona pellucida also occurs in higher numbers. IVF outcome also increases as compared to other processing techniques. GWCF, based on proven clinical success, is therefore invaluable for sperm processing.

Cell Separation↗

Sperm morphology assessment using strict criteria and male fertility under in-vivo conditions of conception.

The clinical significance of sperm morphology assessment according to very strict criteria was determined using semen samples of randomly chosen males from couples not submitted to assist procreation techniques, with a median duration of infertility of 4 years (range 1-17; n = 89). The relationships of sperm morphological properties to the results of standard sperm analysis, including the differentiation of round cells in semen by monoclonal antibodies and semen cultures, the testing of sperm functional capacity in vitro with the standardized sperm-cervical mucus penetration test (SCMPT) and the subsequent pregnancy rate under in-vivo conditions of conception, were evaluated in a prospective study. The quick staining method (DiffQuick(R) stain) for sperm morphology proved to be practical and suitable for routine use. The percentage of normal forms according to strict criteria ranged from 1 to 36%, with a median of 12%. Morphological findings were not markedly related to the medical history, but significant relationships between standard parameters of sperm analysis, in particular the sperm count, the progressive motility and standard sperm morphology, were found. The percentage of normal forms was not significantly associated with the microbial colonization of semen samples but was negatively related to high leukocyte rates. Semen samples with a higher percentage of normal spermatozoa (shown, for example, for >4,> 7 or >=14% normal) were significantly more frequent in cases of an adequate SCMPT. The subsequent pregnancy rate within an observation period of 12 months was 20.2%. The pregnancy rate under in-vivo conditions was significantly higher when semen samples had a better sperm morphology, with significant differences for thresholds at 4, 7 and 14% of strictly normal forms. Although sperm morphology is only one among a multiplicity of factors determining fertility, the results suggest that the evaluation of sperm morphology using strict criteria provides valuable information during basic infertility investigations.

Adult↗

Alteration of cervical mucus by vanguard human spermatozoa.

Movement characteristics of 'vanguard' and 'following' human spermatozoa within cervical mucus were measured using high-speed cinemicrography. The data were applied to a mathematical model of the hydrodynamics of sperm-mucus interaction, using the methods of analysis of covariance and stepwise multiple regression. Vanguard spermatozoa swam more rapidly and more efficiently than did following spermatozoa, although the flagellar beat frequencies and amplitudes of the spermatozoa in the two populations did not differ. This distinction in sperm-mucus hydrodynamics could be due to reduced flagellar thrust and/or increased mucous resistance experienced by the following spermatozoa.

Cervix Mucus↗

[Ovarian follicular maldevelopment in infertile women].

OBJECTIVE: To study the manifestation and outcome of ovarian follicular maldevelopment (FM) in infertile women. METHODS: Among 296 infertile women, 80 cases with FM were taken as study group and 20 with normal ovulation as control. Serial B-ultrasonography, cervical mucus scoring, serum estradiol (E2) assay and laparoscopy, endometrium histology were monitored in both groups. RESULTS: The incidence of FM in infertile women was 27.0%, B-ultrasonography showed many small and obscure outlined follicles in the ovarian. The maximum and mean cervical mucus socres in FM group were < 9 and 7.3 +/- 1.8, significantly lower than those in control (> 10, 13.2 +/- 1.8) (P < 0.01). So were the midcycle serum E2 levels (300 +/- 100 pmol/L vs 900 +/- 400 pmol/L, P < 0.001). No stigma was found in two-third of FM patients and no corpus luteum in about one-third. Proliferative endometrium was shown in 34.0% of the FM group. CONCLUSION: (1) FM with an incidence of 27.0% might be an important cause of infertility. (2) FM results finally in follicular atresia or multiple immature luteinized follicles.

Adult↗

Methods for monitoring follicle maturation decrease; the ovarian hyperstimulation syndrome during gonadotropin treatment.

Therapy with human menopausal gonadotropin and human chorionic gonadotropin (hMG-hCG) has proved effective for induction of ovulation in patients with hypogonadotropic hypogonadism showing anovulation or sterility. However, two important complications, the ovarian hyperstimulation syndrome (OHSS) and multiple gestations, are observed in a few patients treated with these hormones. In the past, follicular maturation was evaluated by the cervical mucus score (CM) and serum estradiol level (E2). But recently, ultrasonographic monitoring (USG) and the measurement of urinary estrogen (E) with a commercial kit of sufficiently high sensitivity have been advocated for the prevention of these complications. In this study, we investigated the relationships between the monitoring methods during hMG-hCG treatment and the rates of the OHSS, ovulation, pregnancy and multiple pregnancy.

Chorionic Gonadotropin↗

Sperm recuperation and cervical insemination in retrograde ejaculation.

Seven infertile patients with retrograde ejaculation, in which spermatozoa could be recuperated from the postejaculation urine, were admitted to a sperm recuperation and cervical insemination program. A noninvasive method for sperm recuperation based on urine alcalinization and serial controls to time masturbation has been used. Insemination has been timed according to BBT charts and cervical mucus characteristics. Pregnancy has been obtained in the seven couples after one to eight treatment cycles.

Cell Separation↗

Differential inhibitory effects of opioids on cigarette smoke, capsaicin and electrically-induced goblet cell secretion in guinea-pig trachea.

1. Goblet cell secretion in guinea-pig airways is under neural control. Opioids have previously been shown to inhibit neurogenic plasma exudation and bronchoconstriction in guinea-pig airways. We have now examined the effects of morphine and opioid peptides on tracheal goblet cell secretion induced by either electrical stimulation of the cervical vagus nerves, exogenous capsaicin, or acute inhalation of cigarette smoke. The degree of goblet cell secretion was determined by a morphometric method and expressed as a mucus score which is inversely related to mucus discharge. 2. Morphine, 1 mg kg-1, completely blocked goblet cell secretion induced by electrical stimulation of the vagus nerves. Morphine also inhibited the response to cigarette smoke given either at a low dose (10 breaths of 1:10 diluted in air), which principally activates cholinergic nerves, or at a high dose (20 breaths of undiluted), which activates capsaicin-sensitive sensory nerves, by 100% and 73% respectively. In contrast, morphine had no significant inhibitory effect on capsaicin-induced goblet cell secretion. The inhibitory effect of morphine was reversed by naloxone. 3. Selective mu- or delta-opioid receptor agonists, [D-Ala2, NMePhe4, Glyol5]enkephalin (DAMGO) or [D-Pen2, D-Pen5]enkephalin (DPDPE) respectively, caused a dose-related inhibition of low dose cigarette smoke-induced goblet cell discharge, with DPDPE more potent than DAMGO. A kappa-receptor agonist, trans-3,4-dichloro-N-methyl-N-(2-(1-pyrollidinyl)cyclohexyl) benzeneacetamine (U-50,488H), had no inhibitory effect. DPDPE had no inhibitory effect on goblet cell secretion induced by exogenous methacholine. 4. DAMGO dose-dependently blocked the response to high dose cigarette smoke with a maximal inhibition of 95% at 2 x 10(-7) mol kg-1. Neither DPDPE nor U-50,488H had any significant inhibitory effect. The increase in goblet cell secretion induced by exogenous substance P was not affected by DAMGO.5. We conclude that opioids inhibit neurally-mediated goblet cell secretion via actions at prejunctional delta and mu-receptors on cholinergic nerves and at mu-receptors on sensory nerve endings, and that capsaicin activation of sensory nerves is via a different mechanism from that of electrical or cigarette smoke activation.

Animals↗

[The treatment of sperm antibodies with corticosteroids].

Seven women whose infertility was thought to be due to sperm antibodies alone were treated with either high or low doses of corticosteroids. During the treatment period the significance of the sperm-cervical mucus contact test was evaluated as a monitor for sperm antibodies. Five women became pregnant after steroid therapy and it was considered to be an effective method of treating infertility due to sperm antibodies since none of the patients complained or suffered from any side-effects.

Antibodies↗

Methods of monitoring menstrual function in field studies: attitudes of working women.

This study was designed to determine the attitudes and compliance of working women toward methods being evaluated for use in the assessment of the effects of toxicants on reproductive potential. Women such as the highly motivated fertility patients and nurses, who are typically familiar with the methods and procedures of fertility assessment and the value of medical research, have been used to validate such methods in a clinical setting. However, the attitudes of a general working female population toward these methods are unknown. Nine participants were selected on the bases, in part, of not seeking fertility assistance, working full-time but not in the medical field, and having less than one year of college education. Attitudes were also evaluated for 193 non-participating women to whom the procedures had been verbally described. Participants measured basal body temperature and salivary and vaginal mucous electrical resistance, evaluated cervical mucus manually (CME), and collected the first morning urine for two menstrual cycles. Blood, saliva, and transvaginal ultrasonograms (US) were obtained at a fertility clinic 6 to 9 days per cycle. Participants brought urine to the laboratory every 3 days. All participants performed all methods. Participants were paid $400; nonparticipants were not compensated. Only 3% of the respondents objected to the proposed methods: principally to CME, US, and giving blood samples. No respondent perceived the study as unimportant.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

Residual embryos in failed embryo transfer.

One hundred thirteen embryo transfers (ETs) were performed in an in vitro fertilization and ET program of which residual or extruded embryos were found in 17 transfers (15%). Residual or extruded embryos are those embryos found outside the uterine cavity, either at the cervical os, on the vaginal speculum, or remaining in the catheter after an ET. Patients were divided into two groups according to the method of inspection for residual embryos. In group I, microscopic visualization of the transfer catheter alone revealed that 9 of the 67 transfers (13.4%) had 17 residual embryos either at the catheter tip or adherent to mucus on the side of the catheter. In group II inspection of the catheter and cervical wash revealed that 8 of 46 ETs (17.4%) had 15 residual embryos that failed to be transferred during the initial attempt. Six of these 15 (40%) were found in the cervical wash medium. Thus, failure of the proper placement of embryos at the time of transfer may occur frequently. Evaluation of only the transfer catheter may result in a significant underestimation of the problem.

Animals↗