Mid-piece length of spermatozoa in different cattle breeds and its relationship to fertility.

Recently positive correlation has been found between oxygen consumption (ZO2) in bull spermatozoa and non-return rates and concluded that an increase in ZO2, characteristic of the freeze/thaw process, was possibly associated with mitochondrial membrane damage during this procedure: alternatively, sperm may be hyperactivated through the capacitation-like effects of freezing/thawing. We speculated that the morphology of spermatozoa may be associated with their rate of ZO2 and fertility: for example, sperm mid-piece length where mitochondria are located. Such a relationship has not been investigated before, particularly in context of commercial cattle breeding programmes and bull fertility characteristics. Sperm biometry was performed on ejaculates obtained from 34 bulls representing six breeds: Holstein (yearlings and mature), Friesian, Belgian Blue, Aberdeen Angus, Charolais and Limousin. Five ejaculates were collected from every bull and from each sample a semen smear was fixed and stained with eosin/nigrosin: the mid-piece length of 40 sperm with normal morphology was measured in every sample. Data were analysed by breed, age and within each bull. Significant differences (p<0.01) between ejaculates in 9/34 bulls was found, as well as differences (p<0.001) between individual bulls within the same breed. The average mid-piece length for Aberdeen Angus was 13.35 microm, for Belgian Blues and Limousin around 13.8 microm and for Charolais 13.68 microm: for dairy breeds such as Holstein and Friesian it was about 13.4 microm. The mean value of mid-piece length for breed was compared with their 49 day non-return rate; a negative correlation (r = -0.53) was found in black and white dairy breeds.

Integrin cell adhesion molecules on human spermatozoa.

The expression of integrin cell adhesion molecules (ITG-CAMs) by human ejaculated spermatozoa (fresh, capacitated and acrosome reacted) was evaluated by immunocytochemical, immunofluorescence and cell-ELIS methods, using monoclonal antibodies against alpha-6 and beta-3 subunits. Both the subunits were expressed on the acrosome region in fresh spermatozoa and post acrosomal region after acrosome reaction induced by calcium ionophore. The spermatozoa of the fertile men showed significantly (P < 0.001) higher expression of alpha-6 and beta-3 ITG subunits than the subfertile men. The percentage of spermatozoa reacting with alpha-6 and beta-3 mAbs increased significantly after the loss of acrosome when compared with fresh spermatozoa. Moreover, 35-40% of spermatozoa with normal shape and none of the spermatozoa with pathological shape showed a positive reaction. The quantitative analysis carried out by ELISA suggests that the levels of these ITG subunits decreased significantly (P < 0.001) in the subfertile subjects when compared with the fertile and the difference was more for alpha-6 than the beta-3. Hence our result suggests that alpha-6 subunit may be used as a clinical marker to evaluate the sperm quality in men.

Resazurin reduction test to evaluate semen quality.

The resazurin test (REZ TEST) was performed on semen samples obtained from 75 untreated subfertile and 10 pregnancy confirmed fertile males. The results were compared with other sperm parameters to assess the quality of semen. The highest correlation of REZ grade was with motile sperm (r = 0.90, P < 0.001), sperm viability (r = 0.80, P < 0.001) and sperm concentration (r = 0.71, P < 0.001). The REZ grade indicated significant difference in the rate of positive tests between samples from fertile males and patients exhibiting abnormal semen parameters. The RES TEST had a positive predictive value of 93.75% for a progressives motile sperm concentration of > 20 x 106/ml and a negative predictive power of 88.7% for the same variable at a criterion value of 10 x 106/ml. It is suggested that REZ TEST can be performed with a relatively small volume of semen, it is easy to perform and requires no technical equipments.

Spermicidal activity of Magainins: in vitro and in vivo studies.

Spermicidal efficacy of two synthetic antimicrobial peptides, viz. Magainin-A (Mag-A) and Magainin-G (Mag-G), have been studied under in vitro condition using rat and human spermatozoa. The data showed that sperm motility was inhibited with various concentrations of Magainins at different intervals ranging from 20 to 480 seconds as compared to controls. The minimum concentration of Mag-A required to immobilize spermatozoa (40-60 x 10(6)/ml of saline) within 20 seconds was found to be 100 micrograms in rat and 200 micrograms in human. The action of Mags on sperm motility was observed to be dose-dependent. The activity of Mag-A was significantly (P < 0.05) higher than Mag-G (P < 0.05). The in vivo studies in rats indicated that Mag-A when applied intravaginally, on the day of estrus, at the dose level of 200 micrograms, for five consecutive estrous cycles, induced no abnormalities either in the length of estrous cycle or in morphology of vaginal epithelial cells. When Mag-A was applied intravaginally before mating on the proestrusestrus day, complete arrest of sperm motility with 200 micrograms concentration was observed and none of these treated females conceived after the mating. In view of these facts, it is suggested that Mag-A, having anti-bacterial, anti-viral, anti-fungal and spermicidal activities, could be a potent vaginal contraceptive.

Lymphocytes--their possible endocrine role in the regulation of fertility.

PROBLEM: Peptide secretion by peripheral lymphocytes obtained from fertile and infertile women. METHOD: LH, FSH, and prolactin were estimated in the serum and culture supernatants obtained from Con-A stimulated human lymphocytes by RIA. RESULTS: The levels of LH, FSH and prolactin were low in the culture supernatants obtained from lymphocytes of infertile women, when compared with proven fertile women. CONCLUSION: The results indicated impairment in endocrine functions of lymphocytes obtained from infertile women. This impairment in lymphocyte function could be one of the causes of infertility in these women.

Early pregnancy factor (EPF) as a marker for detecting subclinical embryonic loss in clomiphene citrate-treated women.

PROBLEM: A discrepancy exists between the apparently normal ovulation and the pregnancy rates in women treated with clomiphene citrate (CC). Our previous studies have indicated that immuno-suppressive "early pregnancy factor" (EPF) is a novel marker to detect subclinical embryonic loss in infertile women. METHOD: In the present study EPF was used as a marker to detect subclinical embryonic loss in women treated with CC with/without gonadotropins. In some of the women treated with CC, conception was assisted by artificial insemination with husband's semen (AIH). RESULTS: Our results have indicated that fertilization occurred (EPF + ve) in 47.7% (52/109) of women treated with CC with/without gonadotropins; 13.46% (7/52) retained the fetus and continued pregnancy till full term, whereas 78.9% (41/52) did not retain the fetuses. In the group where after stimulation, conception was assisted by AIH, fertilization was observed in 38.24% (26/68), retention in 11.54% (3/26) but subclinical embryonic loss was observed in 80.8% (21/26) cases. CONCLUSION: Thus, our results have indicated that subclinical embryonic loss may account for some of the discrepancy observed between the apparently normal ovulation and the pregnancy rates in women treated with clomiphene citrate.

Early pregnancy factor as a marker for assessing embryonic viability in threatened and missed abortions.

It is now well recognized that the presence of early pregnancy factor (EPF) can signify the occurrence of fertilization, continuation of pregnancy and the existence of a viable embryo. With this in view, a study was undertaken to observe the potential of EPF as a marker in assessing embryo viability in cases complicated with vaginal bleeding during early pregnancy. The results indicated that the sensitivity of EPF as a marker in predicting threatened or missed abortion was 78.9% and the specificity 95.6%. The positive predictive value was observed to be 93.8% and the negative predictive value 84.6%. Our studies have shown that since EPF is present in viable but absent in non-viable pregnancies, it could be a useful marker of prognostic value in threatened abortions.

Early pregnancy factor (EPF) as a marker for the diagnosis of subclinical embryonic loss.

The validation of EPF as a possible correlate of early fertilization has made it possible to study and detect fertilization of the ovum in normal fertile women (during the luteal phase) and also in women with infertility, where the fertilization of the ovum may not be affected but there may be impairment in early embryonic development which results in early embryo loss or subclinical embryo loss. Our results have suggested that using EPF as a marker, we could detect subclinical embryonic loss in 57.8% of the infertile women where more than one menstrual cycle was studied and the blood was collected 4-7 days after ovulation. After the missed period, 80% of the patients who were negative for EPF but positive for hCG had spontaneous abortions. It would be interesting to study how EPF behaves as a marker, to detect subclinical embryonic loss in diverse pathological situations such as recurrent abortions, parental age and translocation carrier parents.

Detection of immunosuppressive activity in human seminal plasma by an immunobioassay.

Recent evidence has shown that under in-vitro conditions human seminal plasma can interfere directly or indirectly with the function of cells of the immune system. It is however, questionable whether the results generated in vitro can be related directly to in-vivo activity. We have therefore standardized an in-vivo immunobioassay to detect the immunosuppressive property of human seminal plasma using adoptive transfer of contact sensitivity to a specific antigen such as dinitrofluorobenzene. Our results indicate that when sensitized lymphoid cells were incubated in vitro with human seminal plasma, their ability to transfer the delayed hypersensitivity in non-sensitized mice was suppressed or inhibited in comparison with the controls. The percentage suppression varied with different samples but the results indicate clearly that the immunosuppressive properties of human seminal plasma can be demonstrated using an in-vivo immunoassay.

Presence of sperm antibodies and association with viscosity of semen.

Semen characteristics were studied in 96 men from an in-vitro fertilization and embryo transfer programme. Along with the routine semen analysis, the presence of sperm antibodies in seminal plasma was measured by an ELISA technique. Antibodies to spermatozoa (IgA and/or IgG) were present in 19 cases and 15 of these (78.95%) had abnormally high viscosity, often associated with a high percentage of particulate debris and an increased number of morphologically abnormal spermatozoa.

Detection of early pregnancy factor-like activity in women with gestational trophoblastic tumors.

The presence of immunosuppressive early pregnancy factor (EPF) in the maternal serum has so far been associated with gestation. Its presence in the serum of women with gestational trophoblastic tumors was investigated. The results indicate that while EPF activity was detected in the serum of women with choriocarcinoma, no such activity was detected in the serum of women with hydatidiform mole, leading to the novel use of EPF as a marker to distinguish these two clinical situations. Results of the experiments also suggest that EPF moiety present in the maternal serum during pregnancy may be of different molecular entity than that present in the serum of women with choriocarcinoma.

Isolation and partial characterization of early pregnancy factor (EPF) from human pregnancy serum.

The isolation and partial characterization of an immunosuppressive early pregnancy factor (EPF) present in the serum of pregnant women, between 3 and 8 weeks of gestation, is described. EPF was purified using ion-exchange chromatography, affinity chromatography and HPLC gel permeation techniques. A homogeneous, active fraction containing a single polypeptide of Mr 21,000 was obtained. This 21 kDa polypeptide appears to represent the major form of rosette-inhibiting active material present in maternal serum during early human gestation.