Sodium alginate potentiates antioxidants, cryoprotection and antibacterial activities of egg yolk extender during semen cryopreservation in buffalo.

The study was conducted to determine effects of sodium alginate on sperm during cryopreservation. Each ejaculate (n = 20) of five buffalo bulls (3-5 years) were divided into six equal fractions and diluted using egg yolk based extender supplemented with different concentrations of sodium alginate and cryopreserved. Frozen-thawed semen samples were evaluated using the CASA, hypo-osmotic swelling test, cervical mucus penetration capacity test, and chlortetracycline fluorescence assay (CTC). Phosphorylation of tyrosine containing proteins and malondialdehyde concentration of sperm membrane were evaluated using immunoblotting and thiobarbituric acid reactive substance assay respectively. The semen extender's anioxidative capacities were estimated by conducting 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays, metal chelating capacity by assessing ferrozine and antibacterial capacity using agar plate methods. Supplementation of sodium alginate in extender improved sperm longevity, plasma membrane integrity as well as capacity to transit through the cervical mucus. Supplementation of extender with sodium alginate minimises the phase transition of sperm membranes and phosphorylation of tyrosine containing proteins during cryopreservation. Malondialdehyde concentration of sperm was less in sodium alginate-treated sperm as compared with control samples. The results indicated that sodium alginate increased antioxidant capacity of semen extender. Supplementation with sodium alginate also improved the metal chelating capacity and antibacterial properties of the extender. In conclusion, supplementation of extender with sodium alginate enhances free radical scavenging, metal reduction and chelating capacities to protect sperm during cryopreservation.

Nanoparticle-mediated drug delivery to treat infections in the female reproductive tract: evaluation of experimental systems and the potential for mathematical modeling.

A variety of drug-delivery platforms have been employed to deliver therapeutic agents across cervicovaginal mucus (CVM) and the vaginal mucosa, offering the capability to increase the longevity and retention of active agents to treat infections of the female reproductive tract (FRT). Nanoparticles (NPs) have been shown to improve retention, diffusion, and cell-specific targeting via specific surface modifications, relative to other delivery platforms. In particular, polymeric NPs represent a promising option that has shown improved distribution through the CVM. These NPs are typically fabricated from nontoxic, non-inflammatory, US Food and Drug Administration-approved polymers that improve biocompatibility. This review summarizes recent experimental studies that have evaluated NP transport in the FRT, and highlights research areas that more thoroughly and efficiently inform polymeric NP design, including mathematical modeling. An overview of the in vitro, ex vivo, and in vivo NP studies conducted to date - whereby transport parameters are determined, extrapolated, and validated - is presented first. The impact of different NP design features on transport through the FRT is summarized, and gaps that exist due to the limitations of iterative experimentation alone are identified. The potential of mathematical modeling to complement the characterization and evaluation of diffusion and transport of delivery vehicles and active agents through the CVM and mucosa is discussed. Lastly, potential advancements combining experimental and mathematical knowledge are suggested to inform next-generation NP designs, such that infections in the FRT may be more effectively treated.

A comparative randomized trial on the impact of two low-dose oral contraceptives on ovarian activity, cervical permeability, and endometrial receptivity.

In a double-blind randomized study, the suppression of ovarian activity and anti-conceptive effects on the cervix and endometrium were assessed during administration of two low-dose monophasic oral contraceptives (20 micrograms ethinyl estradiol [EE], 500 micrograms norethisterone--Eve 20 [Grünenthal, Aachen, Germany]; 20 micrograms EE, 150 micrograms desogestrel --Lovelle [Organon, Munich, Germany]). One hundred eighteen healthy women (ages: 18-35 years) were studied in 10 investigation centers during medication with either Eve 20 (n = 59) or Lovelle (n = 59). During three treatment cycles, ovarian activity was evaluated by sonographic determination of follicle-like structures (FLS) and by simultaneous assessment of serum endocrine profiles (gonadotropins LH and FSH, ovarian steroids estradiol [E2] and progesterone [P]). While on either treatment, no ovarian activity (as judged by no FLS and/or reduced sex steroid levels) was found in 90.8% (Eve 20) and 97.2% (Lovelle) of all investigated cycles. Follicular activity or cyst formation were detected in 18 of 173 cycles (Eve 20) and in 5 of 175 cycles (Lovelle), respectively. Gonadotropin levels were suppressed (LH < 6 IU/L, FSH < 8 IU/L) in most treatment cycles (Eve 20 76.6% vs. Lovelle: 84.8%). Serum E2 concentrations exceeding 0.1 nmol/L indicated residual follicular activity in 19.3% (Eve 20) versus 12.2% (Lovelle) of all cycles. An estimated by serum P levels over 5 nmol/L, ovulation had presumably occurred in 4.1% (Eve 20) versus 2.9% (Lovelle) of treatment cycles. However, when the sonographical and endocrinological data were combined, no ovulation was documented in any pill cycle. The quality and quantity of the cervical mucus was found to be minimal in the majority of women. Moreover, the endometrial layer was determined to be low by ultrasound during most pill cycles, indicating equally strong suppressive effects on endometrial receptivity by the two contraceptives. These observations suggest that ovarian activity is suppressed in the majority of cycles during use of low-dose contraceptives. This effect may mainly be medicated by pronounced suppression of serum gonadotropin levels. Strong anti-conceptive effects of these formulations on both cervical permeability and endometrial receptivity are additional factors ensuring the contraceptive efficacy of these formulations.

PIP: The impact of two low-dose monophasic oral contraceptives (OCs) on suppression of ovarian activity, cervical permeability, and endometrial receptivity was investigated in a randomized double-blind study involving 118 healthy women 18-35 years of age recruited from 10 study centers in Germany. 59 women received Eve (20 mcg of ethinyl estradiol and 500 mcg of norethisterone) and 59 were given Lovelle (20 mcg of ethinyl estradiol and 150 mcg of desogestrel) for a total of 3 cycles. No ovarian activity, as assessed by sonographic determinations of follicle-like structures and serum endocrine profiles, was detected in 90.8% of cycles of Eve users and 97.2% of cycles in the Lovelle group. Follicular activity or cyst formation was found in 18 of 173 cycles of Eve users and 5 of 175 cycles of Lovelle users. Gonadotropin levels were suppressed (luteinizing hormone under 6 IU/L and follicle-stimulating hormone less than 8 IU/L) in 76.6% of treatment cycles in the Eve group and 84.8% of cycles in the Lovelle group. Serum estradiol concentrations exceeding 0.1 nmol/L, indicative of follicular activity, were recorded in 19.3% of cycles of Eve users and 12.2% of cycles in the Lovelle group. Although serum progesterone levels were over 5 nmol/L in 4.1% of cycles in the Eve group and 2.9% of those in the Lovelle group, consolidation of sonographic and endocrinologic data failed to document ovulation in any treatment cycles. The quantity and quality of cervical mucus was minimal in most women in both groups. Finally, the endometrial layer was determined to be low by ultrasonography during most pill cycles, confirming the OCs' equally strong suppressive effects on endometrial receptivity.

Quantitative post-coital test: sperm counts in cervical mucus after enzymatic liquefaction.

The post-coital test involves direct microscopic examination of sperm number and motility in cervical mucus. The results depend on the quality of the mucus and the distribution of spermatozoa within the sample. To progress from such qualitative data to quantitative measurements of the spermatozoa present in post-coital mucus, we have developed methods to measure sperm concentrations in enzymatically liquefied post-coital cervical mucus. The mucus score and sperm motility were measured prior to mucus liquefaction, and, together with sperm concentration, they allowed the calculation of the total number of motile spermatozoa present. A combination of bromelin and glycosidases proved to be more efficient in achieving reliable mucus liquefaction than treatment with bromelin alone, and was used to liquefy a series of 36 post-coital test samples. Total sperm numbers ranged between 19 x 10(3) and 16.8 x 10(6). Of the samples, 75% contained < 3 x 10(6) spermatozoa, and 39% contained < 1 x 10(6) spermatozoa. Sperm motility was very high in these samples, except for a distinct subset of samples (19%) in which the total sperm motility was markedly decreased ( < 20%). The measurement of sperm concentration in liquefied cervical mucus will help to determine normal values for the post-coital test, and to estimate the number of motile spermatozoa reaching the upper female genital tract.

The activity of candidate virucidal agents, low pH and genital secretions against HIV-1 in vitro.

The effect of low pH, normally present in the female genital tract, on HIV viability was examined. HIV is more acid stable than previously reported with no substantial reduction in infectivity occurring until pH levels are reduced below 4.5. The virucidal activity of 3 topical spermicides and chlorhexidine was assessed in vitro using previously established and newly modified assay systems. None of the agents tested had a selectivity index (SI) greater than 5.2. Semen and cervical secretions were assessed for their ability to inhibit HIV-1. While no virucidal effect was found in the latter, seminal fluid was found to have significant activity against HIV-1 and a SI of approximately 50.

Release characteristics, ovarian activity and menstrual bleeding pattern with a single contraceptive implant releasing 3-ketodesogestrel.

The properties of a single contraceptive subdermal implant releasing 3-ketodesogestrel were assessed in fifteen women over twelve months. Serum levels of 3-ketodesogestrel were monitored regularly following insertion and after removal. The mean serum level of 3-ketodesogestrel was 245 pg/ml after 72 h (steady state) and 176 pg/ml after twelve months. All volunteers demonstrated ovulation inhibition throughout the study. Transient oestradiol peaks occurred during the study. No luteal activity was noted. The cervical mucus was rapidly rendered hostile to sperm migration. Two women withdrew from the study during the first six months for medical reasons. Both volunteers cited bleeding irregularity as the main cause, one complaining of oligomenorrhoea, the other of prolonged bleeding/spotting episodes. A small but significant increase in weight was noted during the study period.

PIP: 15 sterilized women participated in a clinical trial of the implant Implanon (Organon), a single ethylene vinyl acetate rod containing 60 mg 3-ketodesogestrel (3-KDG), the metabolite of desogestrel. The rod is 40 mm long, 2 mm in diameter and is packaged in its inserter. In this trial the implants were treated to simulate the 2nd year of use. The study subjects underwent intensive hormone and ultrasound monitoring for 72 hours after insertion, twice weekly for 6 weeks and at 6-month intervals. 13 women completed 6 months, 7 completed 12 months, and 5 continued the trial 24 months. There were no complications related to insertion or removal. 3-KDG levels rose to a steady state of 245 pg/ml by 72 hours, then fell to a mean of 17 pg/ml at 12 months. 90 pg/ml of 3-KDG is the critical serum level for anovulation. After removal, 3-KDG declined to 54 pg/ml in 3 days. Follicle development tended toward small follicles or those larger than 10 mm. There was no luteal activity, and LH, FSH and progesterone remained in the follicular phase range. Estradiol levels were not low enough to risk osteoporosis. There was no significant change in serum sex hormone binding globulin. Systolic blood pressure decreased significantly at 12 months; mean weight gain was 3.7 kg (range from loss of 4 kg to gain of 22 kg); a variety of bleeding irregularities were recorded by individual women.

Studies on the nature of prolactin-like immunoreactivity in bromelin-treated cervical mucus.

Prolactin has been reported to be present in cervical mucus at concentrations higher than those found in blood. Our initial findings appeared to confirm this and the material fulfilled criteria of validity generally applied when an immunoassay is employed on a new biological matrix, i.e. parallelism and chromatographic identity. Further experiments demonstrated that prolactin concentrations in cervical mucus were less than 40 mU/L and the prolactin-like immunoreactivity originally detected was due to the action of the enzyme bromelin which was used to liquefy the mucus. Bromelin has a similar molecular weight to prolactin and appeared to digest prolactin tracer and reduce its ability to bind antiserum in a manner paralleling the effect of adding pituitary prolactin.

A changed responsiveness to oestrogen in ewes with clover disease.

When clover-infertile ewes are subsequently exposed to non-oestrogenic pasture, they have a reduced fertilization rate, due to an inability to store spermatozoa in the cervix, and the cervical mucus has a reduced spinnbarkeit, caused by a slower response to oestrogenic stimulation. Vaginal cell keratinization and oestrous behaviour occurred more slowly after treatment of affected ewes with oestrogen. Other changes in affected ewes suggest that phyto-oestrogens have permanent mild differentiating effects on adults. Sexual behaviour is masculinized, the cervix takes on a uterine-like appearance and the genital tract becomes permanently oestrogenized. The manner in which these changes relate to the altered responsiveness to oestrogen remains to be clarified.

Bromelin for liquefaction of cervical mucus in sperm antibody testing: its effect on spermagglutinating immunoglobulin G.

Incubation of cervical mucus with the proteolytic enzyme bromelin is known to result in complete liquefaction of the gel. This effect has been used in the detection spermagglutinating antibodies in cervical mucus. However, the well-known papain-like effect of bromelin on human immunoglobulin G (IgG) might be expected to lead to an underestimation of the sperm antibody activity in cervical mucus. This possibility was investigated by studying the effect of bromelin on known amounts of purified IgG from four spermagglutinating sera. Serologic testing of bromelin-treated samples of cervical mucus mixed with purified IgG revealed no effect on spermagglutinin titers, whereas a significant effect was observed on purified IgG alone. Inert proteins in cervical mucus seem to protect the antibodies against degradation, apparently by substrate competition. Liquefaction of cervical mucus by bromelin seems to be a better basis for the assessment of the sperm antibody content of the mucus than extraction procedures, which have been shown to give a deficient yield.

[Clinical and laboratory evaluation of prolonged administration of microdoses of d-Norgestrel]. / Surveillance clinique et biologique durant l'administration prolongeé d'une faible dose de d-norgestrel

PIP: Experience with a small dose of d-Norgestrel as an oral contraceptive in 60 patients is reported. 336 cycles were observed. 2 pregnancies occurred, after 1 month and 9 months treatment; in both cases the patients had not taken the medication regularly. Side effects included spotting (50%), menstrual cycle irregularity (40%), headache, increased appetite, venous congestion, and raised blood pressure (1 patient each). 33 patients left the study. Serum FSH and LH levels were studied in 3 patients, vaginal smears in 38 patients, cervical mucus in 10 patients, and endometrial biopsy in 9 patients. Hepatic function was not affected in the 10 patients studied.^ieng