The variable morphological response of the endometrium to endogenous hormones in the naturally cycling patients reflects either an unique physiological response to hormones at the target tissue, or an insufficiency in the amount of endogenous hormones at the level of the uterine epithelium, or both. Problems associated with the endogenous hormone response, coupled with the variability observed in the response of women with no ovarian function to exogenous E2 and P4, [5, 51, 6, 52, 53] highlights the need to optimize both the dose and timing of hormone replacement regimes and where possible, to tailor regimes to suit individual patients .
Tissue samples examined from the naturally cycling patients consistently displayed epithelial tissue, a heterogeneity of cellular morphology and a variable morphological response to endogenous hormones. Having previous failed ET attempts, observations of endometrial variability in these patients indicates the importance of attaining a mature 'in-phase' endometrium if implantation is to proceed.
In general, uterodomes appear after D5 of P4 stimulation  and the fact that several patients displayed developing uterodomes prior to this time (D3P) indicated that they would have been fully developed by D5P. However, the presence of fully developed uterodomes on D3P would have placed these patients into the 'patient variability' group where an out-of-phase epithelium can occur with a temporal span of up to 5 days .
The presence of large exposed glands in some of the patients suggested an epithelium under the influence of E2 alone or insufficiently stimulated by P4, thus out-of-phase and indicative of late proliferative endometrium. Other indicators included areas of tissue displaying large, pleiomorphic cells with raised cell borders.
The observation of patients, within the naturally cycling control group, with a hyperplastic (over proliferative/hyperoestrogenic) [46, 47] endometrium demonstrated the often unsuspected endometrial variability in naturally cycling women, adding weight to the argument in support of uterine biopsy and SEM for direct visualisation of endometrial morphology.
Patients recruited into the downregulation cohort displayed a more uniform tissue response to the HRT that followed. The two patients that had biopsies taken after pituitary down-regulation but prior to HRT, indicated that an unstimulated, flat epithelium, resembling the unstimulated epithelium of the postmenopausal uterus, can be seen in ovulating women, and that HRT in a consecutive cycle can restore that epithelium to a mature functional state. It was interesting to note however, that statistical analysis of the morphological characteristics indicated that HRT after downregulation was significantly different from the natural cycle in a number of characteristics. The median score of downregulation plus HRT showed that exogenous hormones had a greater tissue effect than that found with exposure to endogenous hormones.
The D5P biopsy taken from these downregulated patients following hormone stimulation, displayed developing and fully developed uterodomes. As uterodomes are known to occur after 5–7 days of P4 (in a natural cycle) [55, 43] this appearance of uterodomes indicates that the HRT regime given was sufficient to stimulate a previously downregulated, flattened uterine epithelium. All patients had a similar morphological response, with a well stimulated epithelium representative of the mid secretory phase. The presence of developing uterodomes was suggestive of a receptive (or pre receptive) state, which under a progestegenic influence, is indicative of the maturing epithelium required for successful blastocyst implantation. Patients that displayed developing uterodomes at D5P could have gone on to develop them later in the cycle, or may have benefited from HRT dose manipulation.
The variability of tissue observed from menopausal patients can be explained by the climacteric process. During the climacteric period there is a physiological decline in ovarian function, a consequential fall in the secretion of both E2 and P4, and an endometrium that eventually becomes atrophic. Since that atrophic state represents the preserved or 'petrified' cycle that existed when the menopause started, it may have several forms. If the last menstrual cycle was ovulatory and ended with a regular menstrual flow, then a 'simple' atrophy will develop. However, if the last cycle or cycles were anovulatory, or if the proliferative phases were irregular, then the 'petrified' state of the last proliferative phase will be observed .
A dramatic improvement from the menopausal baseline of the 20 epithelial characteristics chosen for the study was observed after 2 cycles of HRT. The baseline biopsies showed only scant areas of an unstimulated epithelium covering the stroma . After HRT, there was an abundance of epithelium which was observed to be similar to that of the mid secretory stage, D19-22, of a functionally receptive epithelium . The microvillous cells were small and uniformly polygonal in shape, had apical cell protrusion and were covered with dense, medium to long microvilli. There was no evidence of cell defect or separation as found in the baseline biopsy. Ciliated cells were moderate to abundant and cilia were tall, upright and covering the entire cell surface . Uterodomes were found fully developed in 10 patients, developing in 3 and absent in 5.
Of the 5 patients with no uterodomes, 3 patients showed little morphological response to HRT indicating that the hormone dose was insufficient to stimulate the epithelium, rather than an inability of the epithelium to respond.
Since patient variability is known to occur with a temporal 5 day 'out-of-phase' period , it is probable that the 24–48 hour window for their appearance either preceded the biopsy date (where biopsy date was D23) or was yet to occur. Since there is no information as to how many cycles of HRT are required before a maximum epithelial response is achieved, it is possible, from the findings of this study, that severely atrophic endometria may need more than 2 months HRT before achieving a standard epithelial response .
Statistical analysis showed that tissue from menopausal patients after HRT differed significantly from natural cycle control patients in 4 of the characteristics observed. However, menopause after HRT differed significantly from downregulation after HRT in 7 of the characteristics observed. When considered with regard to the median scores, the tissue response to HRT after downregulation seems more advanced, with menopausal tissue retaining some of its pre-stimulation characteristics, such as single cilium, less dense microvilli and flattened cells.
Manipulation of the follicular phase in naturally cycling patients did not interfere with the morphological integrity of the secretory epithelium as observed by SEM. Our results also demonstrated that uterodome appearance, abundance and shape did not appear to be compromised by utilizing a short follicular phase cycle.
Seven pregnancies which resulted from this part of the study were achieved using the long follicular phase regime with 5 days of P4 in patients who were assessed by previous biopsy to have optimal epithelium with this regime . Perhaps premature P4, after a short cycle of E2, may accelerate the closure of the nidation window and compromise the chances of successful implantation [41, 42]. This is also indicated by a significant difference in secretion, with tissue from the long follicular cycle being less secretory than that from the short follicular cycle (Table 2).
The function of uterodomes in humans is still unknown but their appearance at, or just prior to the time of implantation suggests they are necessary for the functionality of the secretory epithelium if implantation and pregnancy are to progress. While pregnancies were achieved with ET on D19 (D5P) of a long follicular phase cycle, fully developed uterdomes were observed on both D10 (D3P) and D12 (D5P) of the short follicular phase cycle. This finding again highlights the potential benefit of repeat biopsies to fine tune individual hormonal priming and the role that P4 plays in determining endometrial receptivity.
In artificial cycles maintained with exogenous hormones, uterodomes have been shown to occur around D22 (D8 P) , suggesting that the window of receptivity in artificial cycles, induced by HRT, can be postponed [34, 36, 39]. Studies where sequential sampling was performed during the same cycle of natural or artificial cycles (non ovarian stimulated), the timing of uterdome appearance was found to vary up to 5 days between women . This individual variability could explain our observation of fully developed uterdomes on both days 3 and 5 of P4 in the same patient, although not in the same cycle. However, we did not observe an abundance of uterdomes on day 22 (D8P) . In contrast, previous work has shown that uterodome appearance is dependant on P4 in a study on naturally cycling women where exogenous E2 was not supplemented .
Exogenous hormone adjusted cycles may require several treatment cycles before synchronisation of morphological events occurs in patients having functional ovaries. There is also the possibility of a cumulative affect from both sources of hormones. All naturally cycling patients that took part in the HST study displayed uterdomes in one or more of the 3 cycles examined (natural cycle, short follicular phase, or long follicular phase). Where the patient had an understimulated epithelium in the natural control cycle, exogenous hormones in subsequent cycles restored the morphological characteristics of the endometrium but did not necessarily result in the formation of uterodomes. Where the morphology of the secretory epithelia appeared normal in the natural cycle no further enhancement was noted during the following HST cycles, supporting the findings of Martel et al.,  and Nikas et al., . However, a subsequent cycle of HST did not always preserve the integrity of a secretory epithelium when observed in the natural control cycle. This supports the notion of a possible 'threshold' effect where the number of hormonal treatment cycles prior to ET may impinge on morphological integrity and impact on implantation outcome.
In summary, the only significant difference observed between the length of the follicular cycle ie: between 7 or 14 days of E2 therapy was the level of secretion. Where uterodomes were observed on D3 or D5 of P4 exposure they were absent by D8P suggesting that their appearance at or just prior to the time of implantation is necessary for the functional integrity of the endometrium if pregnancy is to progress. Baseline biopsies and manipulation of the secretory epithelium until uterodomes are observed is thus a useful tool for timing ET transfer.