Comparison of CD9 & CD146 markers in endometrial stromal cells of fertile & infertile females

Background & objectives: CD9 and CD146 are important adhesion molecules that play a role in the implantation of an embryo. This study was undertaken to correlate the expression of these markers in fertile and infertile women's endometrial stromal cells. Methods: Human endometrial stromal cell culture from endometrial biopsies of fertile (n=50) and infertile females (n=50) was performed and primary cell lines were established. Expression of CD9 and CD146 was studied for all the 100 cell lines with the help of flow cytometry. Gene expression of CD9 and CD146 was performed by real-time polymerase chain reaction. Results: There was a significant difference in endometrial stromal cells of fertile and infertile females. Flow cytometric results revealed significantly lower expression of CD9 (P=0.0126) and CD146 (P=0.0006) in the infertile endometrial stromal cells as compared to fertile endometrial stromal cells. These results were comparable with real-time data. Interpretation & conclusions: This study showed that endometrial stromal cells from infertile females had lower expression of adhesion molecules, CD9 and CD146. Our findings suggest that CD9 and CD146 may have a role in infertility. Infertile female's endometrial stromal cells have decreased expression of CD9 and CD146 which can be the cause of infertility related to implantation failure.

Effect of estrogen and progesterone on newly synthesised and released human fallopian tube specific proteins.

Current study was carried out to identify the profile of newly synthesized and released proteins by human fallopian tube (hFT). Results indicated that hFT during menopause synthesised and released only 2-3 proteins as against several proteins ranging from molecular weight (MW) approximately 20 to approximately 130 kD during normal menstrual cycle. In vitro addition of estradiol-17 beta (E2) resulted in synthesis and release of a number of proteins including specific protein of MW 110-130 kD. Addition of progesterone (P) however, led to inhibition of protein synthesis and a combination of E2 and P negated the effect of the latter. An alteration in oviductal secretory protein-profile following addition of E2 in vitro were similar to that observed during normal menstrual cycle.

Volume of semen as a parameter of its quality.

A total of 1655 semen samples obtained from men who underwent initial semen analysis before enrolling for IVF-ET programme were analysed. The semen samples were categorized arbitrarily on the basis of volume as high (> 4.5 ml), normal (1.5-4.5 ml) and low (< 1.5 ml). The semen parameters studied vis-a-vis volume of the sample were viscosity, osmolality, motility, sperm count and morphology. The different volume groups were also compared to the overall quality of the sample. The study showed that high and low volume semen were associated with high incidence of spermatozoa exhibiting subfertile characteristics. Normal volume per se did not indicate the quality of the sample, however high and low volumes were indicators of semen of subfertile quality. There was an inverse correlation between osmolality and volume of semen sample. Thus the parameter 'volume' gives a rough estimate of the quality of the sample.

Relevance of plasma fibrinogen estimation in obstetric complications.

We present a study of fibrinogen levels in 133 patients who were prone to develop disseminated intravascular coagulation as a result of an underlying complication of pregnancy such as abruptio placentae, pregnancy-induced hypertension, missed abortion, septic abortion, intrauterine fetal death, vesicular mole and amniotic fluid embolism. A high incidence of hypofibrinogenemia was found in cases of abruptio placentae (43.9%) and pregnancy-induced hypertension (25%). Hypofibrinogenemia occurred in 10% cases of intrauterine fetal death within 4 weeks of fetal demise. The use of this simple investigation makes possible the diagnosis of hemostatic failure and also helps to guide replacement therapy during the fibrinopenic state. There were 4 maternal deaths and 12 perinatal losses in this study.

Incidence of abnormalities in sperm morphology as an indicator of its fertilizing potential.

A retrospective analysis was carried out to assess the correlation of sperm morphology with the occurrence of fertilization in vitro of human oocytes. Seventy women who were subjected to in vitro fertilization procedure were taken up for this study. The cytological smears of semen were evaluated for the morphological characteristics of spermatozoa. Instances where in vitro fertilization did not occur (group II) had significantly (P less than 0.001) higher morphological abnormalities (head defects) when compared with those where fertilization occurred in vitro (group I). The results indicate that evaluation of sperm morphology with special reference to head abnormalities has diagnostic potential in human in vitro fertilization.

Localization and detection of ovarian follicular fluid protein in follicles of human ovaries.

Human ovarian follicular fluid protein has been partially purified and the active fraction designated as hGF2. Using specific polyclonal antiserum to hGF2, it was observed to be localized immunohistochemically in the granulosa cells of medium but not large follicles of human ovary. The hGF2 levels were estimated by ELISA in serum and follicular fluid of 10 gonadotropin-stimulated women recruited for IVF-ET programme. The results revealed a 3-fold increase in the concentration of hGF2 in follicular fluid compared to that in serum of these patients. These data indicate that the protein is secreted by granulosa cells and plays an important role in the regulation of follicular maturation and ovulation.

Comparison of four different ovarian stimulation protocols in an in vitro fertilisation & embryo transfer programme.

Four different ovarian stimulation protocols were evaluated in an in vitro fertilisation and embryo transfer programme in 208 women (228 treatment cycles). In the rigid protocol (RP), 100 mg of clomiphene citrate (CC) was given from day 3 to day 7 of the menstrual cycle and 300 IU of human menopausal gonadotropin (hMG) was given from day 5 of the menstrual cycle. In the individualised protocol (IP) the same drugs and doses were used as in RP, but the day of initiation of CC depended on the length of the individual's menstrual cycle and hMG was administered from the last day of CC. In the programmed protocol (PP), ovarian function was suppressed with oral contraceptive pills (ethinyl estradiol 30 micrograms and norethisterone 1 mg) started on day 5 of the menstrual cycle for 45 to 70 days. Considering the last day of pill intake as day 0, CC was given for 5 days from day 5 and hMG (300 IU) from day 7. In the alternate day protocol (ADP), 100 mg of CC was administered from day 2 to day 6 and hMG (300 IU) was given on alternate days from day 2 to day 8 or day 10 of the cycle. In all the women, hCG (5000 IU) was administered when the diameter of at least 2 follicles was greater than or equal to 16 mm and estradiol levels were 300 pg/ml/dominant follicle. Patients not showing such a response were not treated further. The cardinal events of IVF-ET such as number of good responders, incidence of oocytes harvested, fertilised and embryos transferred per cycle were compared and it was concluded that the pregnancy rates were highest in women treated by the PP.

Endometrium--an extragonadal source of inhibin.

Using polyclonal antibodies generated against human seminal plasma inhibin (10.5 KDa), immunocytochemical localization was carried out in paraffin embedded tissue sections of human endometrial biopsies obtained at various phases of the menstrual cycle. A positive reaction which indicated the presence of inhibin was characterized by the presence of golden yellow or brown colour in the cytoplasm of epithelial cells that formed the glands as well as the luminal lining. The stromal cells however, showed negative staining. In early proliferative phase, the endometrial glands exhibited weak positive staining for inhibin which gradually increased and was intense in late follicular and early secretory phases. The intensity of the staining although was not diminished in the glandular epithelium in the mid as well as late secretory phases, the number of cells showing positive staining appeared to be reduced. Incubation of endometrial biopsies in vitro with labelled amino acid and immunoprecipitation of newly synthesized protein with specific antibodies to inhibin indicated that endometrium is capable of de novo synthesizing inhibin. The above results suggest that endometrium is an extra ovarian source of inhibin and the possible role of endometrial peptide in sperm fertilizing capabilities as well as in pre and post implantation events is suggested.

Ectopic pregnancy after tubal sterilization.

We report 13 cases of ectopic pregnancy following tubal ligation out of 287 ectopic pregnancies seen during a six year period (1984-1989). These findings suggest that tubal sterilization does not invariably confer infertility. Ectopic pregnancy must not be disregarded in women who have undergone tubal ligation, especially if two or more years have elapsed since the sterilization.

Ultrastructure of spermatozoa & non spermatozoal cells in human semen in genital tract infections.

With a view to ascertain the possible etiology fo the morphological abnormalities seen in spermatozoa of semen from genital tract infections, 16 semen samples were studied. Samples were selected on the basis of each of them having 10-12 pus cells per high power field. Apart from routine semen analysis, the sperms were subjected to electron microscopic studies. Alterations in the ultrastructure of spermatozoa and non-spermatozoal constituents of the infected semen samples were studied. The possible etiology of a wide spectrum of abnormalities in semen found to be associated with genital infection is discussed.