Differential expression of calreticulin, a reticuloplasmin in primate endometrium.

BACKGROUND: To our knowledge, there are no data on hormonal regulation of reticuloplasmins in primate endometrium. We report the presence and modulation of expression of three reticuloplasmins in endometrium of bonnet monkeys (Macaca radiata). METHODS: Receptive and non-receptive endometria obtained from vehicle-treated control and onapristone (antiprogestin)-treated animals, respectively, were compared for differentially expressed proteins by two-dimensional proteomics. Mass spectrometric analysis annotated two such proteins as calreticulin and protein disulfide-isomerase (PDI), known to be molecular chaperones in endoplasmic reticulum. We then investigated if endoplasmin, another reticuloplasmin is also differentially expressed. Expression of these reticuloplasmins was also investigated in the endometriuma during pregnancy in bonnet monkeys. Samples were analysed by immunohistochemistry and western blot (calreticulin in human endometrium), and calreticulin transcript levels in Ishikawa cell line were assessed by real time PCR. RESULTS: Immunohistochemical analysis of the functionalis region of non-receptive endometria in monkeys revealed higher expression of (i) calreticulin (P < 0.01) in glandular epithelium and (ii) PDI in stroma (P < 0.0001), but no change in endoplasmin in stroma or glands, compared with receptive endometria. Protein level of all three reticuloplasmins in the stromal region of endometrial functionalis was higher in pregnant than non-pregnant animals (P < 0.05). Human endometrial calreticulin protein was higher in the estrogen-dominant (proliferative) phase than progesterone-dominant (mid-secretory) phase of the cycle. Calreticulin mRNA in Ishikawa cells is up-regulated by estrogen (P < 0.05 versus control), with a trend towards down-regulation by progesterone. CONCLUSION: Our data suggest that endometrial reticuloplasmins are regulated by hormones and embryonic stimuli in a cell-type specific manner. These novel data open up new lines of investigation for elucidating the mechanisms by which hormones or embryonic stimuli influence the sub-cellular physiology of endometrium.

Protein repertoire of human uterine fluid during the mid-secretory phase of the menstrual cycle.

BACKGROUND: This study is an attempt to construct a repository of polypeptide species in human uterine fluid during the mid-secretory phase of menstrual cycle. This information is essential to generate alternative and less invasive tools for the assessment of uterine functions. METHODS: Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and mass spectrometric analysis were used to resolve and identify the major components of human uterine fluid. RESULTS: Uterine fluid collected during the mid-secretory phase (n = 6) demonstrated ca. 590 polypeptide spots in the linear range of pH 4-7 after 2D PAGE. Mass spectrometric analysis revealed the presence of heavy and light chains of immunoglobulins, alpha-1 anti-trypsin precursor, anti-chymotrypsin precursor, haptoglobin, apolipoprotein A4, apolipoprotein A1 fragment, beta-actin fragment, heat shock protein 27, hemopexin precursor and transferrin precursor. 2D protein profile of fluid collected during the proliferative phase (n = 5) revealed ca. 433 polypeptide spots, of which 279 could be paired with mid-secretory phase protein spots on the basis of their coordinates (isoelectric point and molecular weight) in 2D gels. Apolipoprotein A4, apolipoprotein A1 fragment and alpha-1 anti-trypsin precursor were 2-3-fold more abundant in uterine fluid collected during the mid-secretory phase as compared with that in the proliferative phase. Further, 86 uterine fluid polypeptides were conserved across species, being detected in human, rat and bonnet monkeys. CONCLUSIONS: The molecular repertoire of the mid-secretory phase human uterine fluid, when compared with that of the proliferative phase uterine fluid, is broadened due to differential expression of proteins. Further, some of the mid-secretory phase proteins were conserved across species.

Antiprogestin ZK-98.299 and progesterone display differential binding characteristics in the human myometrial cytosol.

To investigate whether the synthetic progesterone antagonist ZK-98.299 binds to progesterone receptor or also has distinct binding sites, the binding characteristics of ZK-98.299 were compared with those of progesterone in the human myometrial cytosol. [3H]ZK-98.299 and [3H]progesterone showed specific binding in the myometrial cytosol and the binding of each radiolabelled ligand could be displaced with the respective ligand in a dose-response manner. However, while the binding of [3H]progesterone could be completely blocked with progesterone or ZK 98.299, the binding of [3H]ZK-98.299 could not be displaced more than 50%. The non-specific binding of [3H]ZK-98.299 was very high as compared to that of [3H]progesterone. Using [3H]progesterone, the relative binding affinity (RBA) of progesterone was more than that of ZK 98.299, whereas using [3H]ZK-98.299 the RBA of ZK 98.299 exceeded that of progesterone. Treatment of myometrial cytosol with increasing concentrations of -SH-modifying agents (iodoacetamide (IA) 0-10 mM or N-ethylmaleimide (NEM) 0-1000 nM) decreased the binding of progesterone by over 80%, whereas similar treatment did not have appreciable effect on the binding of [3H]ZK-98.299. Although both preformed ligand-receptor complexes were relatively stable in the presence of IA and NEM, the [3H]progesterone-receptor complex was more sensitive as compared to the [3H]ZK-98.299-receptor complex. The addition of 20 mM molybdate in the cytosol had a protective effect against the -SH-modifying agents. [3H]ZK-98.299 and [3H]progesterone-receptor complexes also showed differential stability when incubated at elevated temperatures (25 degrees C and 37 degrees C), [3H]ZK-98.299-binding sites being more thermolabile as compared to [3H]progesterone binding sites. Prior occupation of the receptor by the two ligands gave the complexes the ability to resist an elevated temperature of 25 degrees C. Moreover, molybdate stabilized both the liganded and unoccupied receptors at 25 degrees C. When the ligand-receptor complexes were applied onto a prefocused polyacrylamide gel, the progesterone and ZK-98.299-receptor complexes were resolved and focused at pH 7.2 and 8.4, respectively. The results of this study suggest that although progesterone and ZK-98.299 are mutually competitive for binding to progesterone receptor, ZK-98.299 also has distinct binding sites.

Binding characteristics of progesterone and antiprogestin ZK 98.299 in human endometrial and myometrial cytosol.

The binding of ZK 98.299, a synthetic progesterone antagonist, with human endometrium and myometrium cytosol was studied and compared with that of progesterone. Progesterone showed specific saturable binding to its receptors in both endometrium and myometrium. ZK 98.299 and progesterone were mutually competitive for binding to progesterone receptors; however, the relative binding affinity of ZK 98.299 was 16% that of progesterone. ZK 98.299 exchanged the progesterone-labelled receptor sites. [3H]ZK 98.299 showed specific binding which was linearly related to the cytosol protein concentration. The binding was not saturable at 15 nM of ligand. The binding capacity and binding affinity of ZK 98.299 receptor was less than that of progesterone. Progesterone also partially displaced the binding of [3H]ZK 98.299. This study suggest that ZK 98.299 and progesterone both bind to the same protein. However, whether ZK 98.299 binds to progesterone receptors alone or even to other functionally related sites is not known. It appears that ZK 98.299 when present in higher concentration than progesterone would be an effective receptor ligand.

Rab coupling protein (RCP): a novel target of progesterone action in primate endometrium.

Acquisition of functional receptivity by the endometrium is assumed to be effected by progesterone-dependent expression and repression of several genes during the implantation window in a menstrual cycle. In the present study, we employed differential display (DD) reverse transcription-polymerase chain reaction (RT-PCR) to identify progesterone-dependent gene/gene fragments that are differentially expressed during the peri-implantation phase in receptive and nonreceptive endometria, obtained from fertile and infertile bonnet monkeys respectively. Receptive endometria were obtained from regularly cycling (n=5) fertile female bonnet monkeys. Endometrial nonreceptivity was induced by treating bonnet monkeys with either 2.5 mg (n=5) or 5.0 mg (n=5) onapristone (ZK 98.299), an antiprogestin, on every third day for one cycle. Ovulation, levels of circulatory hormones (estradiol and progesterone) and menstrual cycle length did not change in treated animals; however, endometrial growth was retarded. DD2, one of the differentially expressed cDNA fragments, showed higher representation in nonreceptive endometria than in receptive endometria. The DD2 sequence was found to be homologous to the sequence of the carboxyl terminal region of Rab coupling protein (RCP), a recently discovered protein involved in intracellular vesicular trafficking. To confirm the identity of DD2 as RCP, RT-PCR studies were carried out with a forward primer deduced from the RCP sequence and a reverse primer from the DD2 sequence. The product (DDRCP) obtained, when sequenced, revealed 95% homology with the nucleotide number 1196-1757 of human RCP cDNA. Furthermore, the pattern of DDRCP expression at transcript level was found to be similar to that shown by DD2; that is, it was higher in nonreceptive endometrium. Northern analysis using labeled DD2 or DDRCP cDNA fragments identified two transcripts of 6.0 and 4.0 kb in human endometrium. In situ hybridization studies using digoxigenin-labeled DD2 revealed significantly higher (P < 0.05) localization of endometrial RCP transcripts in the proliferative phase than in the peri-implantation phase in control animals. The localization was also significantly (P < 0.01) higher in peri-implantation-phase endometria from antiprogestin-treated animals than in control animals. These antiprogestin-treated animals, however, did not demonstrate any concomitant increase in the levels of immunoreactive endometrial Rab4 and Rab11 during the peri-implantation phase. A similar pattern of cycle-dependent RCP expression was observed in human endometrial biopsies. Furthermore, significantly higher (P < 0.05) levels of RCP transcripts were detected during the peri-implantation phase in women with unexplained infertility (n=3) than in fertile women (n=3). This is the first report indicating the endometrial expression of RCP and its hormonal regulation.

Testosterone, cortisol, prolactin and bioactive luteinizing hormone in day and night samples of cerebrospinal fluid and serum of male rhesus monkeys.

Testosterone (T), cortisol (C), prolactin (PRL) and bioactive luteinizing hormone (bLH) were found to be normal constituents of the cerebrospinal fluid (CSF) of all the 15 adult male rhesus monkeys studied. The CSF levels of the hormones showed a good correlation with their serum levels. The geometric mean values of circulating levels of T, PRL, bLH in all the animals studied were significantly lower in the samples of the two body fluids collected between 09.00 and 11.00 h as compared with those collected between 21.00 and 23.00 h. C levels were higher during the day as compared with the night samples. This marked difference between the day and night levels of the circulating hormones was not observed in a few individuals which suggests that the diurnal changes in circulating levels of these hormones may not occur as a rule in all rhesus monkeys. The serum:CSF ratios for C, PRL and bLH did not vary significantly between the day and night samples of the body fluids as they did for T. This suggests that T is poorly transferred from the blood to the CSF as compared with the other 3 hormones studied. The possible pathways by which the hormones are transferred into the CSF and the functional significance of their presence in the CSF are discussed.

Endometrial contraception: modulation of molecular determinants of uterine receptivity.

Modulation of endometrial receptivity is a promising approach for fertility regulation since it allows a contraceptive to act specifically at the endometrium. This was corroborated by our previous observations that treatment with low doses of a pure progesterone antagonist (PA, antiprogestin), onapristone (ZK 98299), in bonnet monkeys inhibited fertility by selectively retarding endometrial development, without affecting the hypophyseal-hypothalamic function. In the present study, further investigations, undertaken to analyze the molecular repertoire of a nonreceptive primate endometrium, determined expression of: steroid hormone receptors, i.e. progesterone receptor (PR) and estrogen receptor (ER); cytokines, i.e. leukemia inhibitory factor (LIF): transforming growth factor beta (TGFbeta) and its receptor (TGFbetaR); and cell adhesion molecules, i.e. integrins (alpha(v)beta(3), alpha(1)beta(1)). These studies were conducted during the different phases of the normal menstrual cycle and following treatment with different doses of onapristone (2.5 mg, 5 mg, or 10 mg every third day for one cycle) in bonnet monkeys. The molecules were analysed collectively to explore the possibility of a correlation between expression of these markers and endometrial receptivity and to investigate whether there exists a regulatory link between expression of these molecules under in vivo conditions. Three types of expression patterns of endometrial factors were observed during the peri-implantation period following onapristone treatment: 1) LIF, alpha(v)beta(3), and alpha(1)beta(1) showed significant (P < 0.02) down regulation in glandular epithelium of endometria in animals treated with all three doses of onapristone as compared to the control group. This was indicative of their critical role in the progesterone-driven cascade leading to implantation. 2) PR, TGFbeta, and TGFbetaR remained unaffected in the endometria from 2.5 mg treated animals and showed down regulation in animals treated with 5 and 10 mg onapristone as compared to the control group, thereby suggesting that the expression of these markers may not truely reflect endometrial receptivity per se. However, their facilitatory role in preparing the endometrium for implantation can not be ruled out since continued perturbation in the expression of these molecules may affect endometrial growth, remodelling, and differentiation, which in turn may render the endometrium nonreceptive; 3) ER remained unaltered in endometria of animals rendered infertile with 2.5, 5, and 10 mg onapristone. This observation indirectly suggests that onapristone-induced endometrial changes are mediated via some specific mechanisms. The present study clearly demonstrates that endometrial non-receptivity induced at low doses of onapristone is associated with changes in the expression pattern of specific molecular markers. However, no direct correlation was observed between in vivo expression of TGFbeta, LIF, and integrins, thereby lending support to the concept that there exists redundancy or multiple pathways which regulate implantation events.

Induction of menstruation by antiprogesterone ZK 98.299 in cycling bonnet monkeys.

The antiprogestin ZK 98.299 (11 beta-(4-dimethylaminophenyl)-17 alpha-hydroxy-17 beta-(3-hydroxypropyl)-13 alpha-methyl-4,9-gonadien-3-one) was administered s.c. (30mg/day) to two groups of cycling bonnet monkeys. In Group I (n = 6), it was injected from day 16 to 18 and in Group II (n = 9) from day 21 to 23 of the cycle. Each animal served as its own control and in the pretreatment cycle the vehicle (benzyl benzoate: castor oil, 1:4) was administered. During the treatment cycle of these animals the peak in estradiol levels was observed between days 8 to 11 of the menstrual cycle. In Group I animals, administration of ZK 98.299 induced vaginal bleeding in three of the six animals within two days of its first injection. In the remaining three animals the menstrual cycle length was prolonged. However, in all the six animals a premature drop in serum progesterone levels was observed. On the other hand, in Group II in seven animals with ovulatory treatment cycles, administration of ZK 98.299 induced vaginal bleeding within four days of the first dose and significantly shortened the cycle length. A significant decline in progesterone levels was observed in these animals also. However, in two animals in each group, ZK 98.299 induced vaginal bleeding while the serum progesterone levels were still high. Post-treatment cycles were ovulatory but the cycle length was marginally increased in some animals. In two animals of Group II, in which the treatment cycle turned out to be anovulatory, ZK 98.299 did not induce bleeding and had no effect on serum progesterone levels. This study shows that when administered during the luteal phase ZK 98.299 induces vaginal bleeding and premature luteal regression in bonnet monkeys. However, induction of vaginal bleeding may not be associated with drop in progesterone levels. ZK 98.299, therefore, appears to have potential for fertility control which warrants clinical evaluation.

Treatment with a progesterone antagonist ZK 98.299 delays endometrial development without blocking ovulation in bonnet monkeys.

The effects of an antiprogestin ZK 98.299 (onapristone) on serum levels of estradiol and progesterone, and on the endometrial morphology were studied in adult bonnet monkeys. Twelve animals having menstrual cycles of normal duration (24 to 30 days) were randomly distributed into 4 equal groups. The animals in Group 1 were treated (s.c.) with the vehicle (benzyl benzoate: castor oil, 1:10), and in Groups 2, 3 and 4 with 5 mg, 10 mg, or 20 mg ZK 98.299 once-a-week, respectively. Treatment was initiated on day 1 of the menstrual cycle and each animal in Groups 1, 2 and 3 was treated for two consecutive cycles. Since the treatment cycle length of animals in Group 4 was considerably prolonged, they were treated for one menstrual cycle only. Endometrial biopsy was taken around day 20 of the second treatment cycle of first three groups and around day 50 of the 4th group of animals. Treatment with vehicle or 5 mg ZK 98.299 had no significant effect on the menstrual cycle length. Treatment with 10 mg dose had no effect in two animals and prolonged the cycle length in one, whereas, further increase in the dose to 20 mg prolonged the cycle length in all the animals. The duration of menses was generally reduced. Treatment with vehicle or different doses of ZK 98.299 had no effect on ovulation. In animals treated with 5 or 10 mg dose, the pattern of mid cycle rise in serum estradiol levels and progesterone levels during the luteal phase of both treatment cycles were comparable to those of vehicle-treated animals and were suggestive of normal ovulatory cycles. On the other hand, in animals treated with the higher dose (20 mg/week), progesterone levels during the luteal phase were significantly reduced and were indicative of luteal insufficiency. The hormonal data during the treatment period of this group of animals was suggestive of two distinct ovarian cycles indicating that the menstrual bleeding during the treatment period was probably very scanty. Treatment with ZK 98.299 impaired the endometrial development in a dose-dependent manner. In vehicle-treated animals, the endometrium had large and tortous glands with secretions. Treatment with ZK 98.299 caused atrophic changes in the glands as well as in the stroma. The height of the epithelial cells was markedly decreased and they became small and inactive. This study, therefore, suggests that treatment with low doses of antiprogestin ZK 98.299 at weekly intervals does not block folliculogenesis or ovulation, but has an inhibitory effect on the endometrium.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of a 10.5 kDa inhibin in the endometrium of bonnet monkey on treatment with an antiprogestin ZK 98.299: an immunocytochemical study.

Immunocytochemical localization of inhibin was carried out in paraffin embedded tissue sections of the control and antiprogestin (ZK 98.299)-treated bonnet monkey endometrium using polyclonal antibodies generated against human seminal plasma inhibin (10.5 kDa). The study shows that administration of low doses (5 mg/week) of antiprogestin results in an increase in the expression of inhibin by the endometrium. Treatment with higher doses (20 mg/week) caused a decrease in the expression. Since treatment with higher doses also caused atropic changes in the endometrium, the decrease in inhibin could be the result of morphological damage to the endometrium rather than the effects of antiprogestin on the expression of inhibin. The potential involvement of endometrial inhibin in the process of nidation is speculated.

Effects of progesterone antagonist ZK 98.299 on early pregnancy and foetal outcome in bonnet monkeys.

The antiprogestin ZK 98.299 (onapristone) was injected subcutaneously (25 mg/day) for 4 consecutive days during early pregnancy to 8 bonnet monkeys. Retrospective analysis of the data showed that the treatment was initiated between 20 to 30 days after the mid-cycle peak in estradiol levels. In 7 animals, vaginal bleeding was induced within 3.6 +/- 2.7 days (mean +/- S.D.) after the initiation of treatment. However, pregnancy was terminated completely only in 5 animals. In these 5 animals, menstruation was induced 1 to 4 days after the initiation of treatment. Serum progesterone levels also decreased; however, a significant decrease (p less than 0.02) in mean levels was not observed until 5 days after the initiation of treatment. In the other 3 animals, in spite of some drop in serum progesterone levels after the treatment and slight vaginal bleeding in 2 animals, the pregnancy continued. Two animals delivered stillborn foetuses at term. The foetuses weighed 92 and 105 g, which is markedly lower than the normal foetal weight (345 +/- 48 g, n = 6) at birth. The gross appearance of the foetuses was suggestive of recent intrauterine foetal death. In the third animal hysterotomy was performed on day 65; foetus weighed 5 g. Haematoma and blood clots were seen in the placental tissue. This limited data on 8 animals demonstrates that ZK 98.299, at the dose regimen employed, completely terminates early pregnancy in 62% of animals. In the cases in which treatment failed, the pregnancy did not continue unaffected. The endocrine function of the placenta was affected and the foetal growth was retarded.(ABSTRACT TRUNCATED AT 250 WORDS)

Gonadal and pituitary responses to progesterone antagonist ZK 98.299 during the follicular phase of the menstrual cycle in bonnet monkeys.

ZK 98.299 is a potent progesterone antagonist. Its effects on folliculogenesis, bioactive LH, ovulation and menstrual cycle (m.c.) length were studied in adult bonnet monkeys. ZK 98.299 (20 mg/day) was administered s.c., once daily on days 5 to 15 of m.c., to ten animals. The pretreatment m.c. was of 26.5 days (25 to 28 days, mean with 95% confidence limits) and on treatment it was significantly (p less than 0.001) prolonged to 46.9 days (39 to 54 days). The anticipated midcycle rise in estradiol and bioactive LH levels was completely blocked in six and attenuated in three animals during the treatment period. However, the levels did not drop below the early follicular phase levels. In one animal (#90), though the cycle length was prolonged by 5 days the midcycle rise in estradiol and bioactive LH levels was observed during the treatment period and this animal had normal luteal function. Seven animals had delayed ovulation whereas, two had anovulatory treatment cycles. The rise in estradiol and bioactive LH levels, prior to ovulation in the treatment cycles, was compatible with the midcycle rise observed in the pretreatment cycles. Serum progesterone levels during the luteal phase of the treatment cycles were normal in six animals whereas, in two they were indicative of luteal insufficiency. In two animals, the treatment cycles were anovulatory. ZK 98.299 had no effect on the duration of menses. The post-treatment cycles were of normal duration. This study suggests that the administration of ZK 98.299 during the follicular phase blocks estradiol and bioactive LH release and terminates the follicular phase in most of the animals. The follicular phase is reinitiated after the treatment is stopped.

Pharmacokinetics of norethisterone following its oral administration and by spraying it nasally or sublingually to male bonnet monkeys.

Pharmacokinetic parameters of norethisterone (NET) were studied in eight adult male bonnet monkeys following the administration of a single dose of 300 ug. The animals were crossed over between the following three routes of administration: oral ingestion, nasal and sublingual spraying. The results indicate that NET was readily absorbed by all three routes but the Cmax and AUC of NET were significantly greater by the sublingual route. No significant difference in the t 1/2 alpha or t 1/2 beta was observed between the three routes. These findings suggest that the sublingual route offers the possibility of reducing the effective dose of NET, which is widely used for contraceptive purposes.

Effects of intranasal administration of norethisterone on folliculogenesis, cervical mucus, vaginal cytology, endometrial morphology and reproductive-endocrine profile in women.

The effects of intranasal administration of norethisterone (NET) on menstrual cycle length, folliculogenesis, serum levels of estradiol, FSH, LH and progesterone, vaginal cytology, cervical mucus and endometrial morphology were studied in 8 volunteers (age 28 to 39 years, weighing between 46 and 54 kg). The study period comprised 4 consecutive menstrual cycles. In the first cycle (pretreatment cycle), only the vehicle (alcohol, propylene glycol, water; 3:3:4) was sprayed intranasally (100 microliters in each nostril), using a metered nebulizer, once daily from day 3 to the last day of menstrual cycle. In the next two cycles (treatment cycles), NET (300 micrograms/day) was administered once daily, starting from day one of menstrual cycle, between 9 and 10 a.m. The fourth cycle was a post-treatment cycle in which the volunteers were monitored for recovery. Blood samples (about 5 ml each) were collected once daily from day 8 to 24 and thereafter on alternate days until the last day of cycle during all the 4 cycles. Levels of estradiol, FSH, LH and progesterone were measured in the serum samples by radioimmunoassay methods. Cervical mucus samples and vaginal smears were collected once daily starting from day 7 or 8 of each cycle until the mucus was very scanty. Serial pelvic ultrasonography was performed starting from day 7 or 8 until the growing follicle disappeared or throughout the cycle in case a growing follicular cyst was observed. Endometrial aspirates were collected once around day 22 in each cycle and processed for routine histological examination.

Constraints in the development of contraceptives for men.

Considerable efforts have been made to develop a male contraceptive and the studies have provided very useful information in this field. At least five different strategies to develop a male contraceptive have been pursued, namely: inhibition of sperm production, interference with sperm function, interruption of sperm transport, prevention of sperm deposition, and prevention of sperm-egg interaction. Of all these approaches, inhibition of sperm production by using androgens either alone or in combination with progestins have given the most encouraging results. A number of clinical trials substantiate that it is indeed possible to have a reversible, effective and safe hormonal method of contraception. A postmeiotic and epididymal approach to interfere with sperm function or the secretory and metabolic processes of the epididymis is another attractive option of male contraceptive development. A number of chemical compounds have been identified which interfere with sperm function in the epididymis without affecting sperm production, however, the compounds evaluated so far were found to be toxic. Interruption of sperm transport through the vas either by vasectomy or percutaneous intravasal injection of liquids which form cure-in-place plugs is also an attractive option. However, reversibility of the methods is of concern in their wide scale use. The major constraint in developing a long-acting male contraceptive seems to be the need for greater investment for product development. The clinical trials for evaluating the efficacy and safety of the new products and formulations stretch over several years and require enormous financial commitment. Nevertheless, the long-term gain of having a long-acting reversible contraceptive for men is far greater than the financial commitments over few years. Male attitude towards using methods of family planning is much more favourable than originally believed. The pharmaceutical industry as well as the health care providers therefore have a greater responsibility. For early development of a contraceptive for men, it is essential to increase investment and simplify the drug regulatory procedures. The advent of newer technologies coupled with the convergent efforts of scientists will certainly make it possible to have an effective, safe and reversible male contraceptive in the near future.

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.

Ultrasonographic monitoring of ovarian follicles in women using norethisterone for contraception.

OBJECTIVE: The purpose of this study was to examine the effect of intranasal and oral norethisterone (NET) on ovarian folliculogenesis. METHODS: Sixteen healthy, sterilized women with regular menstrual cycles were recruited to the study. NET 300 micrograms per day was administered orally (n = 8) or intranasally (n = 8) for two consecutive menstrual cycles. Serial pelvic ultrasonography was performed to monitor ovarian follicular growth. RESULTS: Ultrasonographic evidence of normal follicular growth and ovulation was observed in 10 cycles whilst 22 cycles were anovulatory. Formation of follicular cysts was seen in 14 cycles, 13 of which were anovulatory and in one ovulation was observed in the opposite ovary. The size of the cysts varied between 27 and 44 mm. The cysts disappeared when NET treatment was discontinued. A positive correlation between cyst size and estradiol levels was observed with intranasal NET in 50% of cyst cycles. In three cycles, although normal follicular growth and endocrine profile were observed, the follicles failed to rupture. These were classified as luteinized unruptured follicles. Immature follicles < 10 mm were seen in six cycles. CONCLUSION: The study showed that NET administered either orally or intranasally evidently disturbs normal follicular growth and rupture.

Gamete intrafallopian transfer: A preliminary experience.

BACKGROUND: After in vitro fertilizationand embryo transfer for tubal infertility, gamete intrafallopian transfer has been introduced for patients with non-tubal infertility. However, the gametes need to be transferred in 2 to 5 minutes and the distance between the operating theatre and tissue culture laboratory delayed its introduction at our hospital. METHODS AND PATIENTS: To overcome this problem we designed a box in which gametes could be stored. Using gametes taken from this box and employing the standard technique, we achieved 5 pregnancies in 39 infertile women. RESULTS: From 41 treatment cycles, 39 women underwent oocyte retrieval. Five pregnancies were achieved of which 4 delivered live births at full term and 1 ended in abortion. Our first gamete intrafallopian transfer baby was born on 6 January 1988. CONCLUSION: The gamete intrafallopian transfer technique can be successfullyadapted for India.

Localization of estrogen and progesterone receptors in the endometrium of common marmosets Callithrix jacchus.

In the present study, changes in the immunohistochemical localization of endometrial estrogen receptor (ER) and progesterone receptor (PR) during various stages of the ovarian cyclicity in common marmoset, have been reported. Ovarian cyclicity was monitored by estimating plasma estradiol and progesterone. During the early follicular phase, weak ER immunolocalization was observed in the endometrial stroma. During the late follicular phase under the influence of rising estradiol levels, stromal ER localization was intense. During the luteal phase, ER localization was absent in the stroma indicating that high concentrations of progesterone suppressed ER. PR localization was not observed in the stroma during the early follicular phase, while weak staining was seen in the stroma during the late follicular phase. PR localization was maximum during the mid luteal phase. However in marmoset, endometrial ER and PR localization was restricted only to the stroma. This unique feature may be due to the characteristic reproductive profile of this nonmenstruating species and needs to be studied further. Thus it can be hypothesized that in the marmoset endometrium, steroid hormone mediated effects possibly occur directly in the stroma and are then transmitted to the epithelium by autocrine/paracrine action of growth factors and cytokines.

Contraceptive potential of an antiprogestin ZK 98.734: reversal of ZK 98.734--induced blockade of folliculogenesis with FSH and LH and their differential effects in bonnet monkeys.

Studies were undertaken in adult bonnet monkeys to investigate whether treatment with an antiprogestin ZK 98.734 at weekly intervals, starting from day one of menstrual cycle, could arrest ovulation and also to determine if ZK 98.734 induced blockade of ovulation could be reversed with gonadotropins. Adult animals have ovulatory menstrual cycles of normal duration were treated at weekly intervals with ZK 98.734 (25 mg/dose, sc, oil base) for 10 consecutive weeks and its effects on serum levels of estradiol, bioactive LH and progesterone, and endometrial histology were investigated. Following treatment with the antiprogestin they were treated with hMG or hFSH alone. Ovulation was blocked during treatment period in all the animals (n = 14). Typical follicular phase rise in estradiol levels was inhibited, mid cycle surge in the levels of bioactive LH was abolished and serum progesterone levels remained below 1 ng/ml throughout the treatment period. However, prolonged treatment had no significant effect on the basal levels of estradiol which were around 50 pg/ml. ZK 98.734 also had no significant effect on cortisol levels. In animals (n = 4) followed for recovery after the last dose, the treatment cycle length was increased to 117.8 + 6.8 days. In three animals the treatment cycles were anovulatory, whereas in one delayed ovulation with luteal insufficiency was observed. The endometrium had become atrophic. Treatment with hMG (Pergonal: 35 I.U. hLH and 35 I.U. hFSH) or hFSH (Metrodin, 35 I.U.) for 7 consecutive days initiated folliculogenesis and the animals ovulated either spontaneously or after a single im injection of hCG (100 I.U.) on day 8 in ZK 98.734 treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)