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.

Regulation of homeobox A10 expression in the primate endometrium by progesterone and embryonic stimuli.

Homeobox A10 (HOXA10), a member of abdominal B subclass of homeobox genes, is responsible for uterine homeosis during development. Intriguingly, in the adult murine uterus, HOXA10 has been demonstrated to play important roles in receptivity, embryo implantation, and decidualization. However, the roles of HOXA10 in the primate endometrium are not known. To gain insights into the roles of HOXA10 in the primate endometrium, its expression was studied in the endometria of bonnet monkey (Macaca radiata) in the receptive phase and also in the endometria of monkeys treated with antiprogestin onapristone (ZK98.299) or in conception cycle where the presence of preimplantation stage blastocyst was verified. In addition, the mRNA expression of HOXA11 and insulin-like growth factor-binding protein 1 (IGFBP1) was evaluated by real-time PCR in these animals.The results revealed that HOXA10 in the luteal phase primate endometrium is differentially expressed in the functionalis and the basalis zones, which is modulated in vivo by progesterone and also by the signals from the incoming embryo suggesting the involvement of HOXA10 in the process of establishment of pregnancy in primates. In addition, the results also demonstrated that the expression of IGFBP1 but not HOXA11 is coregulated with HOXA10 in the endometria of these animals. The pattern of changes in the expression of HOXA10 in response to the two stimuli suggests that endometrial receptivity and implantation not only requires a synchrony of maternal and embryonic signaling on endometrial cells in the primates but there also exists a controlled differential response among the cells of various uterine compartments.

Non-genomic membrane progesterone receptors on human spermatozoa.

Progesterone regulates vital sperm functions such as capacitation and motility; it is also considered as one of the physiological initiators of the acrosome reaction. Progesterone binding and progesterone mediated biological effects are crucial for sperm functions; these are reportedly dysfunctional in a subset of infertile males. Acting through a mechanism independent of transcriptional regulation, the sperm membrane progesterone receptor (PR) demonstrates high structural specificity for the steroid and is unable to interact with progesterone analogs and antiprogestins. At present, the identity of the receptor is unknown; the hormone-receptor interactions are facilitated by albumin and disulphide bonds. Antibodies to the nuclear PR recognize a protein of 55 kDa in sperm lysates that localizes on the acrosomal membrane suggesting the immunological identity of the membrane and the nuclear PR. Decoding the identity of the membrane steroid receptor and understanding the basic cascades of non-genomic mechanisms of progesterone action would be useful in drug designing, targeted towards modifying sperm functions for contraceptive use and for the management of male infertility.

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.

Effects of an antiprogestin onapristone on the endometrium of bonnet monkeys: morphometric and ultrastructural studies.

Our previous studies demonstrated the ability of low doses of antiprogestin ZK 98.299 (onapristone) to inhibit fertility in bonnet monkeys. In the present study cumulative effects of low doses of ZK 98.299 on the endometrial cytoarchitecture of bonnet monkeys were analyzed. Treatment with either the vehicle (n = 3) or onapristone at 2.5 mg (n = 4) or 5.0 mg (n = 3) was initiated on Day 5 of the first menstrual cycle and thereafter repeated every third day for four to seven consecutive cycles. The last treatment cycles were anovulatory in two animals treated with 2.5 mg and all animals treated with 5.0 mg. Endometrial biopsies were collected on Day 8 after the midcycle estradiol peak in ovulatory menstrual cycles and on Day 20 in anovulatory menstrual cycles during the last treatment cycle. Ultrathin sections of the fixed endometrium were stained with toluidine blue for morphometric analysis and uranyl acetate and lead citrate for ultrastructural analysis. The ZK 98.299-treated animals showed a dose-dependent endometrial atrophy as evident by a decrease in the height and diameter of the glands and early signs of compaction in the stroma. Ultrastructural analysis also revealed dose-dependent degenerative changes in the subcellular organelles such as the nucleus, mitochondria, endoplasmic reticulum, lysosomes, and Golgi apparatus. This suggests that long-term treatment with low doses of ZK 98.299 leads to the suppression of estrogen-dependent endometrial proliferation. However, this blockade operates independent of estradiol receptor (ER) and progesterone receptor (PR) concentrations as the expressions of these steroid receptors did not show any significant changes even after prolonged treatment. The study demonstrated an antiestrogenic effect of ZK 98.299 on endometrium after prolonged treatment in bonnet monkeys.

Progesterone receptor as an indicator of sperm function.

Expression of progesterone receptor (PR) localization on spermatozoa was determined in men with normal and abnormal spermiograms. Studies were also carried out to evaluate the potential of PR as a marker of sperm function. Progesterone receptor expression on spermatozoa from men with normozoospermia (n = 8), oligozoospermia (n = 7), asthenozoospermia (n = 8), oligoasthenozoospermia (n = 7), and teratozoospermia (n = 11) was analyzed using an immunocytochemical method with monoclonal antibodies against PR, and flow cytometry using a cell-impermeable fluorescein-tagged progesterone coupled to BSA complex (P-FITC-BSA). Both methods revealed significantly fewer (P < 0.05) PR-positive spermatozoa in men with oligozoospermia, asthenozoospermia, oligoasthenozoospermia, and teratozoospermia compared with men with normozoospermia, thereby suggesting that down-regulation of PR expression in spermatozoa may be one of the causes of male infertility. Spermatozoa from men with normozoospermia (n = 12), oligozoospermia (n = 12), asthenozoospermia (n = 12), oligoasthenozoospermia (n = 9), and teratozoospermia (n = 10) were exposed to low osmotic conditions in the hypoosmotic swelling (HOS) test and then analyzed for PR expression using P-FITC-BSA complex. A significantly higher percentage (P < 0.05) of spermatozoa with physiologically active plasma membrane (HOS+) lacked PR expression (HOS+PR-) in all categories of men with infertility, thereby suggesting that compared to the HOS test, PR expression is a better indicator of sperm function. Furthermore, PR expression in spermatozoa showed a strong (P < 0.05) positive correlation with their ability to undergo an in vitro acrosome reaction. This was observed in all study groups (i.e., normozoospermia, r = 0.8545; oligozoospermia, r = 0.8711; asthenozoospermia, r = 0.7645; oligoasthenozoospermia, r = 0.9003; and teratozoospermia, r = 0.8676). This suggests a potential role for PR in the events leading to the acrosome reaction in sperm.

Expression profiles of endometrial leukemia inhibitory factor, transforming growth factor beta2 (TGFbeta2), and TGFbeta2 receptor in infertile bonnet monkeys.

The expression profiles of leukemia inhibitory factor (LIF), transforming growth factor beta2 (TGFbeta2), and transforming growth factor beta2 receptor (TGFbeta2R) were analyzed during the peri-implantation period in regularly menstruating, fertile bonnet monkeys and in animals in which endometrial nonreceptivity was induced by administering an antiprogestin, onapristone. Based on our previous experiences, a dose of 2.5 or 5 mg of onapristone was administered s.c. every third day during the menstrual cycle, because these dosages impair endometrial development without upsetting the normal gonadal endocrine profiles. Endometrial biopsy specimens were collected during the proliferative phase (estradiol levels about 200 pg/ml, n = 5) and peri-implantation period (Day 8 after midcycle peak in estradiol levels, n = 5) from normal ovulatory animals and during the peri-implantation period from onapristone-treated animals (n = 10). The biopsy specimens were processed to determine the expression patterns of LIF, TGFbeta2, and TGFbeta2R by immunohistochemical and reverse transcription-polymerase chain reaction (RT-PCR) methods. Levels of both protein and mRNA for LIF, TGFbeta2, and TGFbeta2R (analyzed by immunohistochemistry and RT-PCR, respectively) were greater in the endometrial samples collected during the peri-implantation period compared to samples collected during the proliferative phase in control animals. Treatment with either of the two doses (2.5 or 5 mg) of onapristone caused a significant (P < 0.05) down-regulation in the expression of LIF in the peri-implantation endometria. The endometrial expressions of TGFbeta2 and TGFbeta2R mRNAs were reduced significantly in animals treated with 5 mg of onapristone, but not in those treated with the lower dose. However, immunoreactive TGFbeta2 and TGFbeta2R proteins were significantly (P < 0.05) down-regulated in the endometrial samples from both the 2.5- and 5-mg-treated groups. The alterations observed in the expression patterns of LIF, TGFbeta2, and TGFbeta2R were specific, because the expression levels of epidermal growth factor receptor remained unaffected in the endometria from the treated groups. The present study demonstrates derangement in the expression profiles of LIF, TGFbeta2, and TGFbeta2R during the peri-implantation period in infertile bonnet monkeys. It may be hypothesized that TGFbeta2 function is one of the early steps in the regulation of the progesterone-driven cascade of events leading to endometrial receptivity, and that any aberration in this step may adversely affect the subsequent molecular events (i.e., expression of LIF). These data also suggest that potential aberrations in the functional network of locally produced cytokines and growth factors even may occur in an endometrium exposed to the optimal peripheral hormonal levels.

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.

Detection of progesterone receptor transcript in human spermatozoa.

The present study, to our knowledge, is the first to demonstrate presence of progesterone receptor (PR) transcript in human spermatozoa. The study shows the presence of low copy number PR mRNA in mature human spermatozoa. The PR transcript in spermatozoa was detected by reverse transcriptase-polymerase chain reaction using primers specific for the hormone binding domain and the DNA binding domain of the conventional uterine PR. Further, the cDNA sequence of the partial PR transcript from spermatozoa was found to be identical to the region spanning nucleotides 2694 to 3230 of the conventional PR full-length cDNA sequence. This study also indirectly suggests that the PR protein indeed is an intrinsic sperm protein and is not acquired through proteinaceous secretions of accessory reproductive organs.

Leukaemia inhibitory factor in the endometrium of the common marmoset Callithrix jacchus: localization, expression and hormonal regulation.

In the present study, changes in the immunohistochemical localization of leukaemia inhibitory factor (LIF) in the endometrium during various phases of ovarian cyclicity of the common marmoset have been reported. LIF was absent during the early and late follicular phases. LIF was observed mainly in the cytoplasm of the endometrial glands during the early luteal phase, reached maximum intensity during the mid-luteal phase and declined again during late luteal phase. In-situ hybridization also showed a similar cyclic pattern in the expression of LIF. Stromal cells only showed signals for LIF during the mid-luteal phase. In ovariectomized marmosets, graded dosages of oestradiol alone failed to induce the appearance of LIF protein. Progesterone treatment following oestradiol priming, however, induced distinct glandular localization of LIF, indicating that LIF is a progesterone-dependent protein. Thus endometrial LIF is under maternal control and is secreted in response to the increased progesterone concentrations in circulation. It is possible that high concentrations of LIF during mid-luteal phase may prepare the endometrium for blastocyst implantation in marmosets.

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.

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.

Variations in seminal parameters over a 12-month period in captive bonnet monkeys.

Semen samples were collected from adult fertile bonnet monkeys twice a month by penile electroejaculation for twelve consecutive months. Various parameters like semen volume, weight of ejaculate and coagulum, sperm count, sperm motility, sperm morphology, and functional parameters e.g. plasma membrane integrity,in vitro nuclear chromatin decondensation and acrosomal status were evaluated to assess within and between animal variations. Effects of seasonality, if any, on quantity and quality of semen were also studied. Considerable intra- and inter-individual variations in the geometric mean values were observed for semen volume, weights of ejaculate and coagulum, and sperm counts during the study period. On the other hand, sperm motility, morphology, and functional parameters showed less within and between animal variations. Results on motility, morphology, and functional parameters indicated that good semen quality was maintained throughout the year. Various routine and functional parameters did not show any annual variations. The diurnal rhythmicity in circulatory testosterone levels was observed throughout the year. The study shows lack of seasonality in exocrine and endocrine testicular functions and further suggests that motility, morphology, and functional parameters are better indicators of semen quality in captive bonnet monkeys.

Increased male responsibility and participation: a key to improving the reproductive health.

PIP: This article examines the role of male responsibility and participation in the enhancement of reproductive health in India. Men are recognized to be responsible for the large proportion of reproductive ill health suffered by their female partners. Lack of knowledge, nonavailability of acceptable contraceptives and lack of services with quality of care deter men from sharing the responsibility in reproductive health matters. Misinformation regarding male sexuality and limited availability of scientific data contributed men's less involvement in reproductive health. Thus, various strategies are implemented to increase men's awareness of reproductive health and the accessibility of products and services. These strategies include: 1) increasing contraceptive options for men; 2) supporting women's contraceptive use; 3) improving sexual behavior and safe sex practices; and 4) narrowing the gender gap for better fertility control. Moreover, extensive research is required in order to understand men's perceptions and needs about fertility regulation and sexual behavior as well as services development.^ieng

India's needs and priorities in fertility regulation research.

PIP: This article evaluates the needs and priorities of India in fertility regulation research. There are multivariate factors that affect the perceptions of people about fertility regulation in India. The socioeconomic status, level of education and religious beliefs have strong influences on their decision on the family size, spacing between children and also the sex of the children. Moreover, a large number of people are either quite ignorant of or cannot afford to avail with the technologies and services for controlling their fertility. These factors generally perpetuate high fertility, which strongly affect the infant and maternal mortality, and eventually the quality of life both for the present and the future generation. The International Conference on Population and Development Program of Action has provided India with an alternative to develop newer methods of fertility regulation so as to widen contraceptive choices. The objective of research in fertility regulation should ensure the fertility control of couples and ability to plan the number and spacing of children. Research in fertility regulation should be aimed at expanding contraceptive choices by improving the accessibility and acceptability of good quality contraceptive products and services. It would necessitate: improving the quality of care; increasing awareness about sexuality and fertility regulation; empowering women; and increasing male-female partnership in sharing responsibilities for family planning and parenthood.^ieng

Contraceptive research and development during the fifty years of independence in India: achievements and desired goal.

PIP: This paper examines the contributions of scientists in the field of contraceptive research and development during the 50 years of independence in India. The empowerment of couples in controlling their fertility by choice rather than by chance is the main goal of their contraceptive research and development. Their main contribution includes evaluation and amelioration regarding the safety, efficacy, and acceptability of contraceptive methods, and development of new methods for fertility regulation. The contraceptive methods being used include vasectomy, oral contraceptives, and long-acting methods. Moreover, they also proposed a hypothesis with regards to reproductive processes that lead to the development of newer technologies. Thus, their achievements over the past 50 years have enriched the field of contraceptive research and product development.^ieng

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.

Onapristone (ZK 98.299): a potential antiprogestin for endometrial contraception.

OBJECTIVES: The effects of the antiprogestin onapristone (ZK 98.299) on fertility; menstrual cycle length; duration of menses; serum estradiol, progesterone, and cortisol concentrations; and endometrial morphologic features were studied in adult bonnet monkeys. STUDY DESIGN: Five animals were treated subcutaneously with the vehicle and another nine with either 2.5 (n = 4) or 5 mg of onapristone per animal (n = 5). Treatment was initiated on day 5 of the first treatment cycle, and thereafter onapristone was administered every third day for four to seven consecutive cycles. The females were placed with adult males during the periovulatory period, which was assessed by frequent analysis of serum estradiol concentrations. In the final treatment cycle an endometrial biopsy was performed on day 8 after a midcycle estradiol peak in the ovulatory cycle, or around day 20 if the cycle was anovulatory. Blood samples for estradiol, progesterone, and cortisol measurement were collected every third day, except for the periovulatory period when sampling was more frequent. RESULTS: Each of the five animals treated with the vehicle became pregnant: one in the first, three in the second, and one in the third mated cycle, whereas only one of nine treated with onapristone became pregnant. Four animals treated with 2.5 mg of onapristone for 17 cycles and another four treated with a 5 mg dose for 21 cycles did not conceive. In eight animals that did not conceive the first three treatment cycles of six were ovulatory, and in the remaining two animals two cycles of each were ovulatory. During treatment the mean menstrual cycle length was not altered significantly; however, in one it was shortened and in another two it was prolonged. Similarly, the mean duration of menses was not significantly affected, but in some cycles it was reduced. Moreover, there was only slight bleeding in some treatment cycles. Ovulation occurred in 30 of 45 treatment cycles, including the final treatment cycle during which the biopsy was taken, as indicated by serum estradiol and progesterone concentrations. In some of the ovulatory cycles prolonged treatment suppressed luteal activity; however, in the ovulatory cycles the duration of follicular and luteal phases was not significantly affected. In the anovulatory cycles there was a delayed increase in serum estradiol concentrations, suggesting a partial inhibition of folliculogenesis. In treated animals endometrial growth and development was retarded and rendered out of phase. In animals treated with the higher (5 mg) onapristone does the endometrial glands had partially regressed, the secretory activity was blocked, and stromal compaction was evident. The treatment had no significant effect on serum cortisol levels. CONCLUSIONS: This study demonstrates that low-dose onapristone treatment throughout the menstrual cycle prevents pregnancy without disturbing the menstrual cycle and ovulation in the majority of cycles. However, anovulation and luteal insufficiency occurred in some animals during prolonged treatment. The contraceptive effect in the ovulatory cycles seems primarily related to the retardation of endometrial development resulting in the inhibition of endometrial receptivity. It appears likely that a dose or treatment regimen of onapristone that will inhibit endometrial receptivity and prevent implantation without affecting the menstrual cycle even on prolonged treatment could be identified.

PIP: Antiprogestin drugs such as RU 486 (mifepristone), ZK 98.299 (onapristone), and HRP 2000 block progesterone action at the receptor level. They bind to progesterone and glucocorticoid receptors, which leads to an antagonistic instead of an agonistic response. Treatment with these antiprogestins, depending upon the dose, retards endometrial development and impairs gonadotropin release, thereby blocking ovulation. The hypothalamus, pituitary, and endometrium, however, differ in their sensitivity to the antiprogestins, with the endometrium being sensitive to doses which do not seem to affect ovulation. The authors report on their study of the effects of onapristone upon the fertility; menstrual cycle length; duration of menses; serum estradiol, progesterone, and cortisol concentrations; and endometrial morphologic features in adult bonnet monkeys for four-seven consecutive cycles. The study was undertaken to assess the feasibility of using onapristone as a contraceptive agent and to determine its mechanism of action. Onapristone was dissolved in benzyl benzoate and then diluted in castor oil (1:10, vol/vol). 0.5 ml of the vehicle was used to administer each dose subcutaneously. Five monkeys were treated subcutaneously with the vehicle, four monkeys each with 2.5 mg of onapristone, and five each with 5 mg of onapristone. The study found low-dose onapristone treatment throughout the menstrual cycle to prevent pregnancy without disturbing the menstrual cycle and ovulation in the majority of cycles. Anovulation and luteal insufficiency did, however, occur in some animals during prolonged treatment. The contraceptive effect in the ovulatory cycles seems mainly related to the retardation of endometrial development resulting in the inhibition of endometrial receptivity. The authors find it likely that a dose or treatment regimen of onapristone which will inhibit endometrial receptivity and prevent implantation without affecting the menstrual cycle even on prolonged treatment could be identified.

Interaction of newly synthesized antiprogesterone ZK98299 with progesterone receptor from human myometrium.

We have undertaken characterization of binding of the newly synthesized progesterone receptor (PR) antagonist ZK98299 in the cellular fractions of human myometrium. Specific [3H]progesterone and [3H]ZK98299 binding was observed in the cytosol and the nuclear fractions, and could be competitively replaced by either of the steroids in their radioinert form. Although PR occupied by both steroids exhibited nuclear uptake, the extent of nuclear binding was lower with [3H]ZK98299-receptor complexes. The binding of both ligands to PR was a function of the duration of incubation and the protein concentration: it was saturable at 3-6 nM steroids with a dissociation constant of approximately 2 nM. However, the number of ZK98299 binding sites (72 fmoles/mg protein) was lower compared to that of progesterone (322 fmoles/mg protein). The relative binding affinity (RBA) of ZK98299 for the nuclear PR was about 33% that of progesterone. The results of our study suggest that ZK98299 binds to PR in the cytosol and the nuclear fractions. The antiprogestin effects of ZK98299 reported in the literature are PR-mediated and may result from suboptimal nuclear binding/retention of antiprogestin-receptor complexes. Since this study did not involve isolation and study of individual PR isoforms, PR-A and PR-B, the present data should be viewed as representing an average of contributions by the two receptor forms.