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A Quantitative Study of Changes in the Human Corpus Luteum Microvasculature during the Menstrual Cycle¹

^a Departments of Cell Biology, ^b Pathology, and ^c Physiology, Faculty of Medicine, University of Córdoba, 14071 Córdoba, Spain

	ABSTRACT

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Endothelial cells are the most abundant cell type in the corpus luteum (CL), and changes in blood vessels have been proposed to play a pivotal role in CL regression. We have studied quantitatively the changes in the human granulosa-luteal microvasculature in CL of various ages: young (Days 17–19 of the cycle), mature (Days 20–24), old (Days 25–27), early regressing (follicular phase of the following cycle), and late regressing (luteal phase of the following cycle). Blood vessels were identified by immunohistochemical staining for the endothelial cell marker CD34. Because of the anisotropy of blood vessels, both vertical and transverse sections of the granulosa-lutein layer (GLL) were used to estimate relative (volume, surface, and length densities) and absolute (mean cross-sectional area) vascular variables. Full luteinization from young to mature CL was accompanied by a 61% increase in the mean cross-sectional area of vascular profiles and a 52% increase in the mean volume of granulosa-lutein cells, as an estimator of changes in the volume of the GLL. In old and early regressing CL, there was a progressive increase in relative structural vascular variables, due to the shrinkage of the GLL, whereas the mean cross-sectional area of capillaries showed a 53% decrease from mature to old CL. Finally, in late regressing CL, there was a decrease in most relative structural variables, in spite of the increasingly shrunken GLL. The decrease in the capillary diameter found at the late luteal phase most likely leads to a decreased blood flow, and early changes in blood vessels could initiate and/or accelerate CL regression.

	INTRODUCTION

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Blood vessels constitute an important component of the corpus luteum (CL). After follicle rupture at ovulation, newly formed blood vessels invade the avascular granulosa and, in about five days, the CL becomes one of the most highly vascularized organs on a tissue-weight basis [1]. This impressive angiogenic activity is probably due to the release of paracrine angiogenic factors such as prostaglandin E2, transforming growth factor ß1, basic fibroblast growth factor, and vascular endothelial growth factors (VEGFs) by granulosa-luteal cells [2–5]. All these factors are mitogenic for endothelial cells, and, in fact, endothelial cell mitoses are responsible for most of the proliferative activity in the young CL [6–8].

Although the mechanisms regulating capillary growth are not well understood, luteotrophic hormones seem to be the main regulators of CL vascularization. Thus human chorionic gonadotropin (hCG) stimulates the expression of VEGFs in human granulosa cells in culture [5]. In pregnant rats, the increase in the proliferative activity of vascular cells at midpregnancy is induced by placental LH-like hormones [9], whereas in cycling rats the twice-daily prolactin (PRL) surges induced by mating or treatment with exogenous PRL on proestrus increase the number of proliferating endothelial cells in the CL in metestrus [10]. Therefore, some of the effects of luteotrophic hormones are due to their actions on CL microvasculature.

Blood vessels are an integral component of the luteal paracrine system [3, 11–13]. Endothelial cells, as well as microvessel-associated cells (i.e., pericytes), interact with blood-borne immune cells and parenchymal cells. Growth factors and cytokines released by these cells are increasingly implicated in the local control of luteogenesis and luteolysis [12–14]. It is noteworthy that luteogenesis is parallel to the invasion of the granulosa-luteal layer by blood vessels. It is possible that blood-derived factors initiate a series of events that may sequentially regulate luteal cell differentiation.

In addition, reduced vascularization and endothelial cell loss seem to be associated with luteal regression. It is generally agreed that luteal endothelial cells are lost by apoptosis during luteolysis and that endothelial cell injury is an early marker of luteal regression [14–16]. A decrease in the number of endothelial cells has been reported during luteolysis in the sheep [17], and treatment with luteolytic hormones such as prostaglandin F₂ also determines early blood vessel alterations [18]. Vascular apoptotic cells have been found in the sheep [18] and human [7] CL during luteolysis. Depletion of endothelial cells could be a consequence of previous regressive changes in the CL. Conversely, regression of blood vessels may also trigger degenerative changes leading to functional and/or structural luteolysis.

Few studies have been focused on the quantitative changes in the luteal microvasculature during the human CL life span. A decrease in the proportion of luteal volume occupied by blood vessels has been reported in the sheep [19], cow, and human [20, 21] during luteal regression, and several studies have indicated that the number of capillaries was apparently reduced in old human CL [22]. However, the identification of endothelial cells in conventionally stained tissues is difficult because of the small size of capillaries and the occurrence of collapsed and tangentially sectioned capillaries in which the vessel lumen is not apparent.

We have carried out a quantitative study on human CL microvasculature during the CL life span by using specific immunostaining of endothelial cells. Because degenerative changes in blood vessels could be a mechanism of luteolysis, the main focus of this study was the changes in luteal blood vessels during luteal regression.

	MATERIALS AND METHODS

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Tissue Samples

Tissue samples were obtained from the archives of the Department of Pathology from hysterectomized-ovariectomized menstruating women showing normal CL and no clinical history of endocrine pathology. The stage of the menstrual cycle was determined by evaluation of the CL [23] and endometrium [24], and menstrual history. CL were studied only when menstrual history, and luteal and endometrial dates were concordant. The day of ovulation was considered Day 0. CL were classified as young (1–5 days of age, Days 15–19 of the cycle), mature (6–10 days of age, Days 20–24 of the cycle), old (11–13 days of age, Days 25–27 of the cycle), early regressing (14–26 days of age; Days 1–13 of the following cycle), and late regressing (27–40 days of age; Days 14–27 of the following cycle). Five CL for each stage of the cycle were selected. Young CL on Days 15 and 16 were not studied quantitatively because blood vessels had not yet invaded the whole granulosa-lutein layer (GLL).

Immunohistochemistry

Two different endothelial cell markers were assayed. CD31 (PECAM-1) and CD34 antigens are membrane glycoproteins expressed in endothelial cells [25, 26]. Monoclonal antibodies against CD31 (NCL-CD31 clone WM 59) and CD34 (NCL-END clone QBEND/10) were purchased from Novocastra Laboratories Ltd. (Barcelona, Spain). Immunostaining was specific for endothelial cells with both antibodies. However, the intensity of the staining was considerably higher with CD34, which was therefore used in this study. Although CD34 is also expressed in hemopoietic progenitor cells, no immunostained cells other than endothelial were observed in the CL. This antigen has been previously used to study ovarian blood vessels [27]. Blood vessels inside and outside of the CL served as positive controls.

Immunohistochemistry was performed on routinely neutral-buffered formaldehyde-fixed, paraffin-embedded tissues. Five-micrometer-thick sections were placed on poly-L-lysine-coated slides and, after dewaxing and rehydration in graded ethanol series, were incubated in 2% hydrogen peroxide in methanol for 30 min to inhibit endogenous peroxidase and washed in PBS. After being washed in distilled water, sections were immersed in 10 mM citrate buffer and were submitted to antigen retrieval in a microwave oven (2 x 5 min at 700 W). Afterwards, sections were allowed to cool at room temperature, washed in PBS, blocked with normal rabbit serum, and incubated overnight with the primary antibodies (CD34, 1:25 or CD31 prediluted). The sections were then processed according to the avidin-biotin-peroxidase complex (ABC) method according to previously described procedures [10]. Sections were counterstained with hematoxylin. Negative controls were obtained by replacing the first antibody with nonimmune serum.

Stereological Study

Sections immunostained for endothelial cell markers clearly demonstrated that blood vessels in the GLL were anisotropic. These vessels run from the theca-lutein areas toward the central cavity, being therefore parallel to a plane vertical to the external surface of the CL (see Fig. 1). This was particularly evident in those CL showing the classical structure composed of a series of folded lobules surrounding a central cavity. Five CL showing this structure for each of the different phases were selected from a larger series and used for quantitative studies. Vertical sections are more adequate than randomly cut sections because they contain valuable information for CL dating and are commonly used for diagnostic purposes. Six micrographs were taken with a x20 objective from different zones of the CL sections by rotating the section at 60-degree intervals. Thereafter, the embedded slab containing the CL was cut, providing two CL halves that were re-embedded at right angles. After cutting the external tissue and the theca-lutein layer, we obtained transverse sections of the GLL (see Fig. 1). In these sections, blood vessels were cross-sectioned and provided nearly circular profiles from which the blood vessel diameter could be estimated. Six micrographs per CL, taken at different levels of the GLL with the x20 objective, were obtained. Finally, both vertical and transverse sections were printed at a final magnification of x340.

View larger version (123K): [in this window] [in a new window]   FIG. 1. Micrographs from vertical (left panels) and transverse (right panels) sections from mature (A, B), old (C, D), early regressing (E, F), and late regressing (G, H) CL. Some vascular profiles are indicated by arrows. In vertical sections, the boundaries of the GLL (G) have been highlighted by arrowheads. T, Theca lutein areas. Left panels, x35; right panels, x200 (reproduced at 95%).

A frame with 35 cycloid test lines (with orientation distribution proportional to the sine of the angle to the vertical plane [28]) and 70 test points was superimposed on micrographs. This is adequate to estimate surface density in vertical sections from anisotropic structures [28, 29]. In vertical sections, the frame was aligned with the vertical plane, which was easily recognizable in each micrograph, and counting was performed using unbiased counting rules [29]. At the final magnification used, the test line length associated with each test point (l/p) = 22 µm, and the area test associated with each test point (a/p) = 1933 µm².

In vertical sections, the following variables were estimated: 1) volume density of blood vessels (Vv_bv), which corresponds to the proportion of the CL volume occupied by blood vessels: Vv_bv = P_bv/P_GLL, where P_bv is the number of test points on the blood vessel and P_GLL is the number of test points on the GLL; and 2) the surface density of blood vessels (Sv_bv), which corresponds to the surface of blood vessel-parenchyma interface per unit volume of GLL: Sv_bv = 2I_bv/P_GLL (l/p), where I_bv is the number of intersections between test lines and the outer boundary of blood vessels.

On transverse sections, we estimated the following variables: 1) Vv_bv, which was equivalent to that estimated in vertical sections (data from both types of sections were pooled in each CL); 2) the mean cross-sectional area of vascular profiles (_bv) as the quotient between the relative area occupied by vascular profiles (A_Abv = Vv_bv) and the relative number of vascular profiles: Q_Abv = #vascular profiles/P_GLL (a/p), and _bv = A_Abv/Q_Abv = Vv_bv/Q_Abv = P_bv (a/p)/#vascular profiles; 3) the length density of blood vessels (Lv_bv), that is, the length of blood vessels per unit volume of GLL, since for cross-sectioned vessels Lv_bv = Vv_bv/_bv; Lv_bv = #vascular profiles/P_GLL (a/p).

Except for the mean cross-sectional area, the remaining vascular variables (Vv_bv, Sv_bv, Lv_bv) were relative. Therefore, changes in these variables could be due to changes in the blood vessels and/or the reference volume (i.e., the volume of the GLL). The total volume of the human GLL is difficult to obtain because the whole CL is often not available. To obtain an estimate of changes in the volume of the GLL throughout the CL life span, we estimated the changes in the volume of granulosa-lutein cells (GLC). The volume of the GLL is directly proportional to the volume of individual GLC and is valuable in the interpretation of changes in relative variables. For this, two diameters at right angles in one hundred GLC in each phase were measured with a x100 objective and a micrometer eyepiece. The volume of GLC was obtained considering it as ellipsoid.

The means obtained for each variable at the different stages of the CL life span were compared by ANOVA followed by Tukey's test for multiple comparison among means. The 0.05 level was considered significant.

	RESULTS

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Immunostaining with CD34 antibodies was limited to the endothelium of blood vessels. On Day 14 of the cycle, blood vessels were absent in the granulosa layer, but on Day 17 blood vessels had invaded the GLL and had reached the boundary of the central cavity. In young and mature CL, blood vessels showed a wide lumen in transverse sections (Fig. 1, A and B), whereas in old CL they were densely packed and showed a narrow lumen in transverse sections (Fig. 1, C and D). These features increased progressively in early regressing CL through the follicular phase of the following cycle (Fig. 1, E and F). However, in late regressing CL, blood vessels were less abundant (Fig. 1, G and H), although the GLL appeared highly shrunken. Quantitative data are shown in Figures 2 and 3. During luteinization (from young to mature CL), there was a 52% increase in the mean volume of GLC as well a 61% increase in the cross-sectional area of vascular profiles (Fig. 2). During the first steps of CL regression (from mature to old CL), there was an increase in most relative vascular variables (Fig. 3), which was coincident with a 43% decrease in the volume of GLC and with a 53% decrease in the mean cross-sectional area of vascular profiles (Fig. 2). From old to early regressing CL, there was a 69% decrease in the mean volume of GLC, whereas no significant changes were found in most vascular variables at this phase. In late regressing CL, there was a decrease in both the volume and surface densities of blood vessels (Fig. 3), whereas no significant changes were found at this time in the mean volume of GLC (Fig. 2).

View larger version (20K): [in this window] [in a new window]   FIG. 2. Mean volume of GLC and mean cross-sectional area of vascular profiles in young (Y), mature (M), old (O), early regressing (ER), and late regressing (LR) CL. a, p < 0.001 vs. the previous CL type (ANOVA and Tukey's test for n = 5).

View larger version (15K): [in this window] [in a new window]   FIG. 3. Relative vascular variables in young (Y), mature (M), old (O), early regressing (ER), and late regressing (LR) CL. a–d, Significant differences vs. Y, M, O, and ER CL, respectively. Differences were considered significant at the 0.05 level or higher. (ANOVA and Tukey's test for n = 5).

	DISCUSSION

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The CL is a highly vascularized organ. This is necessary to provide steroidogenic cells with the amounts of cholesterol needed for steroidogenesis and for the delivery of progesterone to the general circulation. A close correlation between the blood flow to the luteal ovary and serum progesterone concentrations has been found in previous functional studies [19]. In this study, we analyzed changes in CL microvasculature during the different phases of the CL life span. Both relative (volume, surface, and length densities of blood vessels) and absolute (mean cross-sectional area of vascular profiles) structural vascular variables were estimated.

Relative vascular variables provide valuable information about the degree of vascularization of a tissue and allow comparison among organs of different sizes. The degree of vascularization of the CL could be appreciated better by relative variables (i.e., the volume of blood vessels or the surface of blood vessel-parenchyma interface per unit volume of tissue) than by absolute variables (i.e., the total volume of blood vessels or the total vascular surface per CL), which are dependent on the size of the CL, which shows considerable individual variability. However, changes in relative variables are influenced by changes in the reference volume (i.e., the volume of the GLL) and should be carefully considered. From young to early regressing CL, there was a progressive increase in all relative vascular variables. This was coincident during CL maturation (from young to mature CL) with an increase in the volume of the GLL (estimated by the increase in the mean volume of GLC). The progressive increase in relative vascular variables in spite of the increase in the reference volume, together with the increase in the mean cross-sectional area of vascular profiles and the presence of abundant proliferative activity in vascular luteal cells at the early luteal phase [7,8], was indicative of the existence of capillary growth in the GLL during luteinization. In contrast, during CL regression, the increase in relative vascular variables was coincident with an important decrease in the volume of the GLL. Therefore, changes in blood vessels found at this time were due to the decrease in the reference volume (i.e., the volume of the GLL) and not to an actual growth of capillaries. This was in agreement with the scarcity of proliferating vascular cells during CL regression [6–8]. However, during the luteal phase of the second cycle, the shrinkage of the GLL, which was evident by microscopic examination, was not consistent with the average volume of the GLC, which was not significantly decreased. This can be explained by the fact that, at this time, cell loss seemed to be the main mechanism for the decrease in the volume of the GLL. The remaining GLC became highly vacuolated, probably because of the accumulation of lipid droplets. At this time, most relative vascular variables were decreased in spite of the increasingly shrunken GLL, indicating that there was an actual loss of endothelial cells. This was in agreement with the microscopic observation of less densely packed blood vessels in the GLL and with previous studies reporting a decrease in the number of endothelial cells/pericytes in advanced regressing CL [17].

Although regressive changes in the CL microvasculature occurred in parallel with luteolysis, the CL continued to be a relatively well-vascularized organ even at advanced stages of regression. Previous studies have reported that there was a decrease in the volume density of blood vessels during early regression in the cow [20, 21] and human [20] ovary, and that blood vessels in the human GLL were nearly absent at the late luteal phase [22]. However, in this study, blood vessels were abundant throughout the CL life span, and the volume density of blood vessels, as well as the rest of the relative structural variables, were not decreased up to advanced regression in late regressing CL. The reasons for these differences are probably due to the difficulty of recognizing vascular profiles without specific staining of endothelial cells. This is particularly significant during CL regression, when collapsed capillaries are frequently found and are difficult to distinguish from the rest of the stromal cells in conventionally stained tissues.

During the functional cycle of the CL, changes in the mean cross-sectional area of vascular profiles were parallel to the changes in the volume of the GLC, as well as to the reported changes [30] in serum progesterone concentrations. From Day 25 of the cycle onward, morphological signs of structural luteolysis, including the presence of apoptotic and vacuolated cells and shrinkage of the GLL, were evident. A large decrease in the cross-sectional area of vascular profiles was found at this time in old CL. These changes in blood vessels most likely have important functional consequences and could be determinant in CL regression. Since vascular resistance is inversely proportional to the fourth power of the radius of the vessel, the large decrease in the diameter of blood vessels found at the late luteal phase would lead to a highly increased resistance and, as a consequence, to a large decrease in blood flow. This agrees with functional studies reporting a dramatic decrease in blood flow to the luteal ovary during CL regression in the sheep [19]. This is also in accordance with the decrease in serum progesterone concentrations from mid to late luteal phase [30]. The role of changes in CL microvasculature in initiating and/or accelerating luteolysis is not clear, but blood vessels could be a site of action of luteolytic hormones [19, 31]. Thus, treatment with LH anti-serum or prostaglandin F₂ decreases blood flow to the luteal ovary in the sheep [19]. Furthermore, it has been reported that prostaglandin F₂ also induces the release of oxytocin by luteal cells that leads to vasoconstriction and ischemia, as well as to a decrease in circulating progesterone concentrations [18]. Ischemia could be also a determinant of luteal cell apoptosis. The administration of a luteolytic dose of prostaglandin F₂ in the sheep increases the percentage of apoptotic endothelial cells as early as 12 h after treatment [18]. The first regressive changes in blood vessels found in the present study at the late luteal phase were coincident with the presence of significant numbers of apoptotic cells reported previously [7].

In summary, the early alterations in blood vessels at the late luteal phase suggest that changes in CL microvasculature could initiate, or at least accelerate, CL regression. Furthermore, the presence of relatively abundant blood vessels during the process of CL regression provides the structural basis for an active role of endothelial and/or capillary-associated cells in luteolysis.

	ACKNOWLEDGMENTS

 
The authors are very grateful to J. Molina and P. Cano for their technical assistance.

	FOOTNOTES

 
¹ This work was supported by Grant PB94-0449 from the DGICYT, Spain.

² Correspondence. FAX: 34-57-218288; fi1begac{at}lucano.uco.es

Accepted: November 13, 1998.

Received: September 21, 1998.

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TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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