Immunocytochemical study of progesterone receptors in the human endometrium during the menstrual cycle

Specific mouse monoclonal antibody (alpha PR6) against progesterone receptor was used with an avidin biotin complex technique to localize progesterone receptors in frozen sections of 26 normal cyclic human endometria. Progesterone receptor was detected in the nuclei of epithelial and stromal cells in both the functionalis and basalis layers. In the functionalis, the receptor content increased from the early to the late proliferative phase in both cell components. It remained high in the early secretory phase and decreased in the mid- and late secretory phases, comparatively more rapidly in the epithelium than in the stroma. In the latter, the predecidual cell nuclei were receptor-positive. The menstrual phase endometrium lacked receptors. The basalis was rich in progesterone receptors during the proliferative, early and midsecretory phases in both components and receptor-free during the late secretory and menstrual phases of the cycle. Myometrial smooth muscle cell nuclei contained progesterone receptors, whereas they were absent in endometrial and myometrial vessels. Overall, the epithelial progesterone receptor content seemed to correlate with the endometrial tissue levels of estradiol, possibly reflecting its estrogen sensitivity, whereas the stromal progesterone receptor content during the secretory phase at least, in part, may be constitutively synthetized.     

Variant progesterone receptor mRNAs are co-expressed with the wild-type progesterone receptor mRNA in human endometrium during all phases of the menstrual cycle

Progesterone receptor (PR) variant mRNAs in human endometrium could encode proteins with the potential to alter progesterone action in states of normal and abnormal endometrial development. We have assessed the expression levels of mRNA for the wild-type PR and splice variants of PR mRNA lacking exon 4 (del-4 PR), exon 6 (del-6 PR), exons 4 and 6 (del-4&6 PR), and part of exon 4 (del-p4 PR) or part of exon 6 (del-p6 PR) in the human endometrium throughout menstrual cycle development. Eighty-eight endometrial specimens (47 proliferative, 41 secretory) were collected from patients undergoing hysterectomy for benign gynaecologic causes. Measurements by RT-PCR indicated that mRNAs for wild-type PR, and splice variants del-4 PR, del-6 PR, del-4&6 PR, del-p6 PR, and a novel del-p4 PR were detected in all endometrial specimens throughout the menstrual cycle. Higher levels of wild-type PR and all PR variant mRNAs were found in the early and mid-proliferative endometrial phases than in secretory endometrium. The relative expression of mRNA for all PR variants compared to wild-type PR mRNA, however, did not change through all stages of endometrial development. We, therefore, found no evidence of differential co-expression of the PR variants compared with wild-type PR during normal menstrual development. Future studies will determine if the expression profile of PR variant mRNAs will be different in the endometrium of patients with infertility, recurrent pregnancy loss, or endometrial adenocarcinoma.     

Immunohistochemical assessment of estrogen receptor distribution in the human endometrium throughout the menstrual cycle

Monoclonal antiestrophilin antibodies (H226Sp gamma and H222Sp gamma) were used with an indirect immunoperoxidase technique to localize estrogen receptor in frozen sections of normal human endometrium. Estrogen receptor was detected in the nuclei of the vast majority of epithelial and stromal cells from all early, middle, and late proliferative phase endometria. However, middle and late secretory phase endometria showed a dramatic reduction in the amount of estrogen receptor localized. Specific staining for estrogen receptor in the functionalis of middle and late secretory phase endometria was weak and limited to nuclei of scattered epithelial and stromal cells. In contrast, strong staining for receptor was observed in epithelial cell nuclei of some glands in the basalis. The early secretory phase appeared to be a period of transition from the strong and ubiquitous staining for receptor characteristic of proliferative phase endometria to the weak, focal pattern of estrogen receptor localization characteristic of the functionalis from middle and late secretory endometria. Postmenopausal endometria showed consistently strong nuclear localization of estrogen receptor in epithelial and stromal cells. Myometrial smooth muscle cell nuclei also contained estrogen receptor. However, the endothelial cells of uterine vessels showed no localization of receptor. Specific staining for estrogen receptor was always limited to nuclei; no specific cytoplasmic staining was observed.     

Telomerase activity in the human endometrium throughout the menstrual cycle

In a total of 41 endometrial tissue samples, the relationship between telomerase activity and proliferating cell nuclear antigen (PCNA) labelling index was studied. In samples of endometrium from the proliferative phase of the menstrual cycle, telomerase activity was found in 15 out of 17 cases (88%). Two samples from the early proliferative phase showed negative telomerase activity and a low PCNA labelling index. However, three out of 16 samples of early secretory phase endometrium showed telomerase activity and a PCNA labelling index. In mid- to late secretory phase endometrium, in menopausal endometrium and in decidualized endometrium induced by progesterone neither telomerase activity nor PCNA labelling was found. These results suggest that telomerase activity of the endometrium may be correlated with the proliferative potential of the epithelial cells and that its activity may be regulated by oestrogen.      

Thioredoxin expression in the human endometrium during the menstrual cycle

Human thioredoxin, a cellular disulphide reducing protein, is known to be secreted by some types of cells and to display unique extracellular activities including modulation of cytokine actions and protection of the cell against damage from oxidative stress. This study has been undertaken to investigate the pattern of expression and tissue distribution of thioredoxin in human endometrium during the menstrual cycle. Immunohistochemical studies showed increased thioredoxin immunoreactivity in the glands of the secretory phase compared to those of the proliferative phase. Although the staining of thioredoxin was relatively intense in predecidual stromal cells, the most prominent staining of thioredoxin was present in both glands and stroma of the endometrium in the early secretory phase of the menstrual cycle. Northern hybridization analyses revealed that expression of thioredoxin mRNA in the endometrium of the early secretory phase was approximately 3-fold compared to the other phases of the menstrual cycle, consistent with the results of the immunohistochemical studies. These results suggest that both protein and gene expression of thioredoxin in the endometrium are menstrual cycle phase-specific and highly active in the phase of endometrial differentiation which occurs in preparation for implantation (early secretory phase of the menstrual cycle). Thioredoxin expressed in the early secretory phase of the menstrual cycle may be advantageous for blastocyst implantation.      

Heat shock proteins in human endometrium throughout the menstrual cycle

Human endometrium is a steroid-sensitive tissue and there isevidence that supports the viewpoint that heat shock proteins(HSP) are implicated in the regulation of steroid function.Therefore, in this study we examined the expression of variousmembers of the heat shock family of proteins in the steroid-responsivehuman endometrium. Western blot analysis revealed that the expressionof HSP90 showed minimal changes throughout the menstrual cycle.When normalized to the amount of HSP90, the expression of HSP27,HSP60 and the constitutive form of heat shock protein 70 (HSC70)increased progressively during the late proliferative and earlysecretory phases, and diminished in the mid- to late secretoryand menstrual phases. In contrast, the inducible form of heatshock protein 70 (HSP70) did not undergo these changes. Thecellular and subcellular localizations of these proteins wereexamined in human endometria by immunohistochemical staining.With the exception of HSP70, which was found primarily in theepithelial cells, the immunoreactivity for other heat shockproteins was found in both the stroraa and the epithelium. Immunoreactivityfor HSP27 was found in the lymphoid aggregates within endometrialstroma, and both HSP27 and HSP90 were found in endothelial cells.The immunoreactive heat shock proteins were found in the nucleiand/or cytoplasm of cells. However, no consistent nuclear versuscytoplasmic staining emerged, and such localization was irrespectiveof the site, the cell type or the phase of the menstrual cycle.Our findings show that endometrium has a full complement ofheat shock proteins. The menstrual cycle-dependent changes inthe amounts of heat shock protein suggest regulation by steroidhormones.     

Levels of prostaglandins in human endometrium during the normal menstrual cycle

The levels of prostaglandins in the human endometrium throughout the normal menstrual cycle were measured by bioassay. The identity of prostaglandin F_2α and prostaglandin E₂ in endometrial tissue extracts was established by combined gas chromatography/mass spectrometry.

The levels of prostaglandin F_2α and prostaglandin E₂ are low during the proliferative phase of the cycle (10-25 ng/100 mg tissue). Prostaglandin F_2α rises significantly during the luteal phase to levels of 65-75 ng/100 mg of tissue. Prostaglandin E₂ levels are similar to prostaglandin F_2α levels in the proliferative phase but remain lower during the luteal phase. The prostaglandin E₂ level is highest at menstruation (52 ng/100 mg tissue). The implications of these findings are discussed in relation to the menstrual process and to the steroid hormonal changes during the cycle.

     

Endometriosis: Oestrogen and progesterone receptors in endometriotic tissue and endometrium: comparison of different cycle phases and ages

The levels of oestrogen and progesterone receptors were analysedin 62 endometriotic tissue samples obtained at laparotomy of58 women and in samples of endometrium obtained simultaneouslyfrom 49 of the women. Both ligand techniques and enzyme immunoassayswere used for the assays. The steroid receptor levels were significantlylower in endometriotic tissue than in endometrium, both in cytosoland nuclear fractions. The differences were most pronouncedin follicular phase. In endometrium the oestrogen and progesteronereceptor levels varied during the menstrual cycle, being highestin follicular phase. In endometriotic tissue, the oestrogenreceptor level remained low throughout the cycle. The progesteronereceptor level, determined by immunoassay, increased duringthe menstrual cycle, being higher in luteal than in follicularphase. This cycle phase pattern was found neither in the nuclearprogesterone receptor level nor when the cytosol was assayedwith the ligand technique. In contrast to endometrium, therewere no correlations between patients' age and steroid receptorlevels in endometriotic tissue. The results show that oestrogenand progesterone receptors in endometriotic tissue have a differentpattern during the menstrual cycle than endometrium and thatthe progesterone receptors synthesized are not all biologicallyactive. These results strengthen previous data indicating differencesin the hormonal regulation of the two tissue types.     

Patterns of expression of integrin molecules in human endometrium throughout the menstrual cycle.

A heterogeneous group of cells interact with each other and with the surrounding matrix to form the complex structure of human endometrium. Since the integrin superfamily of molecules is involved in the cell-cell and cell-matrix interactions, this study was designed to screen, in situ, the cellular distribution of CDW49a-f molecules in human endometrium throughout the menstrual cycle. The integrin molecules were localized by immunohistochemistry using monoclonal antibodies. Glandular epithelium expressed all integrin molecules. With the exception of CDW49d (alpha 4 beta 1), surface epithelium also expressed all these molecules. Endothelial cells were positive for all integrin molecules except CDW49a (alpha 1 beta 1). Endometrial lymphoid cells were positively immunostained for CDW49a, d and e (alpha 5 beta 1) and were negative for CDW49b (alpha 2 beta 1), CDW49c (alpha 3 beta 1) and CDW49f (alpha 6 beta 1). Regional differences in the expression of integrin molecules were observed. As compared to the functionalis epithelium, basalis epithelium characteristically exhibited higher expression of CDW49a, d and e. Two integrins in endometrium, CDW49a and d exhibited changes related to the menstrual cycle. CDW49a, which was not expressed in glandular epithelial cells in the proliferative phase, was strongly expressed in these cells after ovulation and its expression was diminished in the late secretory phase. This molecule was not expressed in the stromal cells, however, predecidual cells characteristically expressed this molecule in the late secretory phase. CDW49d was only expressed in the glandular epithelial cells in the mid-proliferative to mid-secretory phases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endothelin synthesis and receptors in human endometrium throughout the normal menstrual cycle

This study was undertaken to investigate the presence of messengerRNA (mRNA) for prepro-endothelin-I (ET-1) and the known receptorsubtypes (ETA and ETB) in human endometrium at different stagesof the menstrual cycle obtained at hysterectomy. Northern blotanalysis revealed expression of ET-1 mRNA in human endometriumduring the normal menstrual cycle. The concentration of ET-1mRNA in endometrial tissue was greater during the menstrualand proliferative phases than during the ovulatory and secretoryphases. Immunoreactive ET-1 was secreted into the medium ofisolated endometrial stromal cells. Oestradiol and progesteronesignificantly attenuated ET-1 release in endometrial stromalcells cultured for 6 days. ETA and ETB mRNA were also presentin endometrial tissue of the normal cycle. The concentrationof ETA receptor mRNA was greater in the proliferative phasethan in the secretory phase, whereas expression of ETB mRNAincreased in menstrual phase. ET-1 significantly increased extracellularaccumulation of cyclic AMP (cAMP), intracellular generationof inositol phosphates and significantly enhanced DNA synthesisin cultured endometrial stromal cells from the proliferativephase. Our results showed that human endometrial cells synthesizedand released ET-1, and contained ETA and ETB receptors whichwere functionally coupled to phosphoinositide breakdown andto adenylate cyclase with the increase of cAMP by ET-1 stimulation.Our findings suggest that ET-1 may have a potential autocrineand/or paracrine function in human endometrial stromal cells.     

Endothelin synthesis and receptors in human endometrium throughout the normal menstrual cycle

This study was undertaken to investigate the presence of messengerRNA (mRNA) for prepro-endothelin-l (ET-1) and the known receptorsubtypes (ET_A and ET_B) in human endometrium at different stagesof the menstrual cycle obtained at hysterectomy. Northern blotanalysis revealed expression of ET-1 mRNA in human endometriumduring the normal menstrual cycle. The concentration of ET-1mRNA in endometrial tissue was greater during the menstrualand proliferative phases than during the ovulatory and secretoryphases. Immunoreactive ET-1 was secreted into the medium ofisolated endometrial stromal cells. Oestradiol and progesteronesignificantly attenuated ET-1 release in endometrial stromalcells cultured for 6 days. ET_A and ET_B mRNA were also presentin endometrial tissue of the normal cycle. The concentrationof ET_A receptor mRNA was greater in the proliferative phasethan in the secretory phase, whereas expression of ET_B mRNAincreased in menstrual phase. ET-1 significantly increased extracellularaccumulation of cyclic AMP (cAMP), intracellular generationof inositol phosphates and significantly enhanced DNA synthesisin cultured endometrial stromal cells from the proliferativephase. Our results showed that human endometrial cells synthesizedand released ET-1, and contained ET_A and ET_B receptors whichwere functionally coupled to phosphoinosttide breakdown andto adenylate cyclase with the increase of cAMP by ET-1 stimulation.Our findings suggest that ET-1 may have a potential autocrineand/or paracrine function in human endometrial stromal cells. cyclic AMP/endothelin-l synthesis/human endometrium/inositol phosphate/receptors     

Erythropoietin and erythropoietin receptor expression in human endometrium throughout the menstrual cycle

Erythropoietin (Epo) is an important regulator of erythropoiesis. Recent studies have demonstrated non-classical sites of Epo and Epo-receptor (Epo-R) expression, suggesting new physiological roles unrelated to erythropoiesis. Other studies have shown that the mouse uterus expresses Epo and its receptor, and produces Epo protein in an estrogen-dependent manner. We therefore hypothesized that Epo is one of the growth factors involved in cyclic endometrial changes. We determined Epo and Epo-R mRNA expression in isolated endometrial epithelial and stromal cells using RT-PCR. While both Epo and Epo-R were detected in all samples of isolated epithelial cells analysed throughout the menstrual cycle, neither one was detected in isolated stromal cells. In addition, using quantitative real-time RT-PCR with the TaqMan detection system, we showed that isolated epithelial cells had higher Epo mRNA levels in the secretory phase than in the proliferative phase. Immunohistochemical analyses revealed that Epo and Epo-R protein expression in glandular epithelial cells was increased during the mid-proliferative phase and was maintained during the late proliferative and the early, mid- and late secretory phases. These findings suggest that Epo may be involved in cyclic proliferation and differentiation of endometrial glandular epithelial cells, acting in an autocrine manner. In addition, we also hypothesize that ovarian steroids may stimulate Epo production in human endometrial glandular epithelial cells.     

Membrane-type matrix metalloproteinases and vascularization in human endometrium during the menstrual cycle

Endometrial angiogenesis is essential for a vascularized receptive endometrium. Previously, we described that membrane type-3 metalloproteinase (MT3-MMP) is associated with endometrial angiogenesis in vitro. The association of MT-MMPs with endometrial angiogenesis in vivo is unknown. Therefore, this study analysed the presence of MT-MMPs in human endometrium and their correlation with neovascularization. RNA/protein expressions of the six MT-MMPs were determined in cultured endometrial cells. Vascularization parameters and MT-MMP expressions in vivo were evaluated by immunohistochemistry in serial endometrium sections. MT1-, MT2-, MT3- and MT4-MMP antigens were expressed in cultured endometrial endothelial cells. MT2-, MT3- and MT4-MMP were expressed by endothelium during the proliferative and secretory phase. Strikingly, these phases showed elevated vascularization, elevated total vascular surface in proliferative phases, elevated number of vessels in proliferative/late secretory phases and increased luminal surface in the proliferative phases. All MT-MMP antigens were expressed in various endometrial cell types in vivo, with decreased levels during the early secretory phase. In conclusion, all MT-MMPs are expressed in endometrium in a cycle-dependent pattern. The vascular expression of MT2-, MT3- and MT4-MMP correlated with angiogenic episodes of the cycle. Since MT2- and MT3-MMP are known to regulate tube formation, these findings support earlier in vitro data on the role of MT3-MMP in endometrial angiogenesis. Additionally, MT2-MMP appears to be associated with endometrial neovascularization also.     

Oestrogen receptor alpha and beta mRNA expression in human endometrium throughout the menstrual cycle.

We examined the localization of oestrogen receptor (ER) beta mRNA in the human endometrium throughout the menstrual cycle using non-radioactive in-situ hybridization with Brigati-tailed oligonucleotides. The findings were compared with those of ERalpha in order to examine the possible biological significance of ERbeta in the human endometrium. Both ERalpha and ERbeta mRNA expression were detected in all major human uterine cell types, including glandular epithelial cells, stromal cells and smooth muscle cells of the uterine wall, at every menstrual cycle stage. However, ERalpha mRNA expression was more prominent than that of ERbeta in all cell types throughout the menstrual cycle. In proliferative phase endometrium, ERalpha mRNA was expressed in both glandular epithelial and stromal cells, while ERbeta mRNA was expressed predominantly in glandular epithelial cells. Although the same pattern was observed in the secretory phase, both the ERalpha and ERbeta mRNA expression was relatively weaker. These results suggest that oestrogenic effects occur predominantly through ERalpha, but that ERbeta may also play a role in the modulation of oestrogenic action, especially on glandular epithelial cells in the human endometrium throughout the menstrual cycle.     

Endothelial cell migratory signal produced by human endometrium during the menstrual cycle.

Human endometrial explants were cultured in a three-dimensional collagen/bovine aortic endothelial cell (BAEC) matrix to measure angiogenic activity, as represented by migration of BAEC towards the explants. The 57 endometrial biopsies were classified by histological appearance into nine stages of the menstrual cycle; five proliferative groupings, three secretory groupings and one menstrual. The BAEC migratory score was taken as the average for 12 explants assayed from each biopsy. The results showed two significant peaks of BAEC migratory activity, one during the early proliferative phase and one during the mid--late proliferative phase. There was a significant drop in the BAEC migratory signal from early--mid-proliferative endometrial explants compared to most other stages of the cycle. The results also show a non-significant rise in BAEC migratory activity in the mid-secretory phase of the cycle. Overall, the results support the concept of two or three different endometrial angiogenic events during the human menstrual cycle, a post-menstrual repair, a mid--late proliferative growth and a lesser mid-secretory activity that may be associated with spiral arteriole growth. Each of these events occurs under different hormonal environments and will need to be investigated separately in terms of local mechanisms controlling angiogenesis.     

Immunohistochemical localization of 17 beta-hydroxysteroid dehydrogenase in the human endometrium during the menstrual cycle.

Immunoaffinity-purified polyclonal anti-rabbit antibody against human placental 17 beta-hydroxysteroid dehydrogenase (17HSD) was used to localize 17HSD in frozen sections of 21 human endometrial tissue specimens, taken at different stages of the menstrual cycle, and in the human placenta. The presence and distribution of estrogen and progesterone receptors were also analyzed in endometrial specimens using commercial immunohistochemical techniques. In addition, 17HSD was localized by immunoelectron microscopy in the endometrium and the placenta. In the endometrium, immunostaining of 17HSD appeared in the cytoplasm of surface epithelial and gland cells during the early and midluteal phase. During the late luteal phase, it gradually disappeared. No immunostaining was observed in the endometrium during the follicular phase of the menstrual cycle. The changes in staining intensity of 17HSD were associated with changes in the concentration of serum progesterone as judged by radioimmunoassay. An apparent inverse correlation between 17HSD expression and the concentrations of estrogen and progesterone receptors was observed. These results strongly support the concept that progesterone induces an increase in the amount of 17HSD, in the glandular and surface epithelial cells of the human endometrium. In the human term placenta, 17HSD immunostaining was detected exclusively in the cytoplasm of syncytiotrophoblasts. In immunoelectron microscopic studies of the endometrium and placenta, 17HSD staining was observed in the cytoplasm and it was associated with cytoplasmic membranes unrelated to the endoplasmic reticulum.     

Distinct subsets of stromal cells confined to unique microenvironments in human endometrium throughout the menstrual cycle.

Human endometrial stroma exhibits rather uniform morphology throughout the endometrium. However, predecidualization develops characteristically around vessels and subsequently around glands and under surface epithelium, demonstrating existence of regional differences among stromal cells. Immunoreactivity of stromal cells in endometrial tissues from various phases of the menstrual cycle, as elucidated by employing monoclonal antibodies to cytokeratin, vimentin, very late antigen-1 (VLA-1), Ber-EP4, and HLA-DR, revealed presence of phenotypically distinct subsets of stromal cells confined to unique microenvironments throughout the menstrual cycle. All stromal cells strongly expressed vimentin and weakly expressed cytokeratin. However, Ber-EP4 positive stromal cells were distinctly confined around glands and to the subluminal regions of the surface epithelium. The intervening stromal cells were Ber-EP4 negative. The HLA-DR positive stromal cells were characteristically present in three different locations: around glands and under surface epithelium, around blood vessels and around HLA-DR positive lymphoid cells. From all antigens studied, only expression of VLA-1 in the stromal cells showed a characteristic change throughout the menstrual cycle. Stromal cells in the proliferative and early secretory phases were VLA-1 negative. However, VLA-1 characteristically developed initially in the HLA-DR positive cells around vessels and then in HLA-DR/Ber-EP4 positive cells around glands and under surface epithelium. Eventually, all stromal cells in the upper functionalis expressed VLA-1 in the late secretory phase of the menstrual cycle. These data underscore a heterogeneity in stromal cells not exemplified by their morphology. Also, they provide a basis for understanding the differences that the stroma exhibits in morphologic and functional differentiation throughout the menstrual cycle.     

Immunolocalization of inhibin and activin subunits in human endometrium across the menstrual cycle

Inhibin/activin alphaC/alphaN and betaA subunits were localized immunohistochemically in the human endometrium throughout the menstrual cycle using an affinity-purified sheep polyclonal antibody raised against the alphaC/alphaN subunit and an affinity-purified rabbit polyclonal antibody raised against the betaA subunit. The betaB subunit was below the level of detection in all human endometrial samples tested. Immunoreactive inhibin alphaC/alphaN subunit was localized in the luminal epithelium, glandular epithelium, stromal tissues and vascular endothelium with no significant variation across the normal menstrual cycle. Immunoreactive betaA subunit, common to inhibin A and activins AA and AB was localized in the luminal and glandular epithelium and in migratory cells while the endometrial stromal cells, decidua, vascular smooth muscle and endothelium were devoid of immunoreactivity. A significant variation of immunoreactive betaA subunit was observed in glandular and luminal epithelium across the normal menstrual cycle. In proliferative endometrium, only a very low level of betaA immunostaining was seen in luminal and glandular epithelium, while the luminal epithelial staining increased significantly in the early secretory phase and remained relatively constant over the rest of the menstrual cycle. A progressive increase in betaA immunoreactivity was observed also in the glandular epithelium during the secretory phase reaching a maximum in the late secretory phases, and decreasing at menstruation. Co-localization studies on serial sections suggested that the migratory cells expressing strong betaA immunoreactivity were macrophages and neutrophils but not eosinophils or mast cells. Thus, cells within the human endometrium are capable of expressing inhibin/activin molecules in vivo. The variation in the pattern of secretion of the betaA subunit across the menstrual cycle suggests that activin peptides may have a physiological role in endometrial function.      

Cycling time trial performance during different phases of the menstrual cycle

Submaximal exercise performance has not previously been assessed in the late follicular phase of the menstrual cycle, which is associated with a pre-ovulatory surge in oestrogen. Therefore, we compared cycling time trial performance during the early follicular (EF), late follicular (LF) and mid-luteal (ML) phase of the menstrual cycle in trained and untrained eumenorrhoeic women who cycled 30 and 15 km, respectively, in a non-fasted state. The women completed the three cycling time trials on a conventional racing bicycle mounted on an air-braked ergometer. We required resting oestrogen to increase by at least twofold above EF phase values in both the LF and ML phases and this resulted in a number of exclusions reducing the sample size of each group. No significant difference was noted in the finishing time between the different menstrual phases in trained (n=5) or untrained (n=8) group, albeit limited by sample size. However, analysis of the combined trained and untrained group data (n=13) revealed a trend for a faster finishing time (P=0.027) in the LF phase compared to the EF phase as 73% of the subjects showed improvements with an average of 5.2±2.9% (or 2.1±1.1 min) in the LF phase (for =0.05 requires P<0.017). Combined group analysis yielded no difference between performance in the EF and ML phase or between the LF and ML phase. Thus, further research is encouraged to confirm the tendency for a faster time trial in the LF phase, which coincides with the pre-ovulatory surge in oestrogen.