Molecular Assessment of the Uterine Milieu during Implantation Window in Humans and Non-human Primates

Geetanjali Sachdeva *, Vishakha Patil , Gracy Rosario , Tanu Parmar , Shruti Nimbkar-Joshi , Rajendra Katkam , Lalita Savardekar ,


Objective: a) To identify the endometrial factors which are directly or indirectly regulated by progesterone in nonconception cycle and also to investigate whether their expression is modulated during early pregnancy in primates and b) To identify the factors which are differentially expressed in endometrial tissues and uterine secretions during the progesterone dominant or mid-secretory phase as compared to the estrogen dominant or proliferative phase in humans Materials and Methods: For objective a six regularly cycling healthy female bonnet monkeys (Macaca radiata) were subcutaneously injected with Onapristone- ZK 98.299 an antiprogestin dissolved in vehicle (benzyl benzoate: castor oil 9:1) at a dose of 5.0 mg starting from day 1 of the menstrual cycle and continued every third day for one cycle. Onapristone treatment rendered the animals infertile or implantation incompetent due to induction of endometrial nonreceptivity. Control animals (n=6) were treated with vehicle alone. Circulatory steroid levels were estimated in animals before and after the treatment using radioimmunoassays (Sachdeva et al 2001; Patil et al 2005). Endometrial biopsies were collected from both onapristone-treated and vehicle-treated animals on day 8 estradiol peak. Immunohistochemistry and reverse tranh1ase polymerase chain reaction were used to investigate whether some of the select factors were differentially expressed after the blockade of optimal progesterone action on endometrium in bonnet monkeys. Differential display reverse tranh1ase polymerase chain reaction (DDRTPCR) and 2D proteomics approaches were also used to identify the factors which were differentially expressed in the endometrium of implantation incompetent bonnet monkeys compared to vehicle treated control animals. To investigate whether the expression of some of these factors is altered during pregnancy endometrial samples were collected from another group of animals on day 6 of pregnancy (approximately equivalent to day 8 post estradiol peak). Towards this regularly cycling female bonnet monkeys (n=6) with normal hormonal profiles (peak estradiol levels- 300-600 pg/ml; progesterone levels (3-6 ng/ml) were mated with males of proven fertility for six continuous days starting from two days prior to the expected estradiol peak. Pre-implantation factor (PIF) in the sera was used as a surrogate marker of pregnancy (Rosario et al 2005a). The control group included nine PIF negative animals. For objective b regularly cycling women (21-35 years) of proven fertility with a history of at least one live birth were enrolled in the study. Ovulation was monitored by serial ultrasonography (USG) to ascertain the follicular collapse. Endometrial tissue and uterine fluid samples were collected from women on day 6 post-ovulation (in mid-secretory phase) or on day 2-3 prior to ovulation (in proliferative phase). 2D proteomics and immunoblot analysis were used to identify the factors which were differentially expressed during the progesterone dominant phase as compared to the proliferative phase in human endometrial tissues and uterine fluid samples. Results: Candidate factor approach revealed differential expression of several cytokines such as interleukin 1 beta interleukin 6 transforming growth factor beta leukemia inhibitory factor (Sachdeva et al 2001) and cell adhesion molecules like alpha v and beta 3 integrin (Puri et al 2000) in the mid-secretory phase endometria of antiprogestin treated bonnet monkeys as compared to that of control animals. This suggested that progesterone directly or indirectly regulates the expression of these factors. Interestingly expression of interleukin 6 transforming growth factor beta in endometrium was significantly higher in the endometria of pregnant animals as compared to that in nonpregnant animals (Rosario et al 2005b) whereas the expression of leukemia inhibitory factor did not alter significantly during early stages of pregnancy. Integrins alpha v and beta 3 also showed cell type specific increase in endometrium during early stages of pregnancy (Nimbkar-Joshi et al unpublished). These studies indicated that progesterone priming during nonconception cycle leads to increase in the expressions of endometrial TGF beta LIF interleukin 6 integrins and these factors probably facilitate endometrial preparation for implantation. Expressions of some of these factors are further modulated during early stages of pregnancy. Functional genomics approaches such as differential display RTPCR demonstrated up regulation of Rab Coupling Protein (RCP) in the endometria of antiprogestin treated animals as compared to control animals (Patil et al 2005). Interestingly RCP is known to be involved in the intracellular trafficking of integrins. There was no concomitant increase in the expressions of Rab4 and Rab11- proteins known to interact with RCP suggesting impairment in the expression of specific components of intracellular trafficking pathways. These studies suggested that the blockade of progesterone action in endometrium may alter intracellular trafficking and this in turn could be responsible for the altered distribution of cell surface molecules such as integrins on endometrium. This could be one of the reasons for the incompetence of endometrium for implantation in antiprogestin treated animals. Further 2D proteomics coupled with MALDI-TOF-TOF analysis revealed differential expression of two reticuloplasmins- endoplasmic reticulum resident proteins such as calreticulin and protein disulfide isomerase in bonnet monkeys rendered infertile with antiprogestin. Interestingly calreticulin was also found to be less abundant in the 2D endometrial tissue protein map of mid-secretory phase as compared to that of the proliferative phase in humans (Parmar et al 2008a). This suggested that the expression of calreticulin is downregulated in endometrial tissues during progesterone dominant phase as compared to estrogen dominant phase Interestingly expressions of these two proteins were also increased in endometrium during very early stages of pregnancy. It may be mentioned here that endometrial estradiol receptor alpha an estrogen regulated gene also showed increased expression during early stages of pregnancy (Rosario et al 2008). This suggested that the expression of these proteins is positively regulated by estradiol in vivo. Our in vitro studies also suggested that estradiol positively regulates the expression of calreticulin whereas progesterone is somewhat inhibitory to the expression of calreticulin. In addition to calreticulin ? chain of fibrinogen adenylate kinase isoenzyme 5 transferrin annexin V alpha-1-antitrypsin (AAT) creatine kinase and peroxidoxin 6 were also found to be differentially expressed in endometrium during the progesterone dominant phase as compared to that in the estrogen dominant phase in humans. Further similar studies on uterine fluid samples revealed higher expression of AAT and apolipoproteins during the progesterone dominant phase or mid-secretory phase as compared to that in the proliferative phase of cycle in healthy fertile regularly cycling women (Parmar et al 2008b). Conclusion: These studies collectively led to identification of several factors in endometrium which are either positively or negatively regulated by progesterone. This knowledge will help in the construction of progesterone regulated functional networks which can be targeted for contraception or for infertility management.