WO2002044716A1 - Methode de controle de l'etat de l'uterus au moyen de cellules tueuses naturelles de l'uterus - Google Patents

Methode de controle de l'etat de l'uterus au moyen de cellules tueuses naturelles de l'uterus Download PDF

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WO2002044716A1
WO2002044716A1 PCT/CA2001/001699 CA0101699W WO0244716A1 WO 2002044716 A1 WO2002044716 A1 WO 2002044716A1 CA 0101699 W CA0101699 W CA 0101699W WO 0244716 A1 WO0244716 A1 WO 0244716A1
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uterine
cells
lymphocytes
adhesion
unk
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PCT/CA2001/001699
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English (en)
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Barbara Anne Croy
Sharon S Evans
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University Of Guelph
Health Research, Inc
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Priority claimed from CA002345478A external-priority patent/CA2345478A1/fr
Application filed by University Of Guelph, Health Research, Inc filed Critical University Of Guelph
Priority to AU2002223348A priority Critical patent/AU2002223348A1/en
Publication of WO2002044716A1 publication Critical patent/WO2002044716A1/fr
Priority to US10/279,884 priority patent/US7094561B2/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5076—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells

Definitions

  • the present invention relates to methods of monitoring uterine status to determine the suitability for a successful pregnancy. More specifically, the present invention relates to methods of assessing or monitoring the luteal phase of the menstrual cycle and/or the first half of pregnancy by assessing the adhesion of human lymphocytes to uterine or lymphoid tissues. BACKGROUND OF THE INVENTION
  • Transient granulated lymphocytes are described in the pregnant uteri of >20 species. 1 In women and mice, these cells are Natural Killer (NK) cells and their activation/maturation depends upon uterine decidualization rather than presence of conceptuses. 2,3 The life history and functions of uterine NK (uNK) cells are more fully known in rodents than women. In women, uNK cells are most frequent in first trimester, representing over 70% of the nucleated bone marrow-derived cells in decidual cell suspensions. Human data also suggest that uNK cells are distinctive, tissue based cells. Most circulating human NK cells are CD16 + CD56 d ⁇ m ; uNK cells are CD16 " CD56 bright .
  • NK Natural Killer
  • the minor circulating CD56 bri9ht subset preferentially expresses (95%) very high levels of L-selectin, 5 a molecule central to initiation of extravasation. Fewer CD56 d,m circulating cells (24%) express L-selection and at a much lower surface density. 5
  • the two major NK cells functions, target cell lysis and cytokine production, may be displayed separately or dually by single cells. 6
  • Human uNK cells display both functions in vitro 7'9 but their in vivo functions are undefined. Many current studies of human uNK function address interactions with trophoblast.
  • IFN- ⁇ Interferon-gamma
  • NK cells The anomalies were endothelial cell hypertrophy and damage in mesometrially- positioned uterine vessels, lack of uterine arteriole wall and lumen changes indicative of pregnancy and hypocellularity of decidua. Absence of lytic NK cell function does not explain these results, thus, a cytokine deficiency hypothesis was pursued.
  • Interferon-gamma IFN- ⁇ is the prototypic cytokine product of NK cells.
  • IFN- ⁇ is an induced molecule known to regulate expression of >1000 genes, many of which are expressed by vascular and decidual tissues. 19,20 IFN- ⁇ is expressed in human and murine uteri during normal gestation 21"23 but many authors regard IFN- ⁇ as detrimental to pregnancy.
  • mice Reproductive cycles of mice differ to human by virtual absence of a luteal phase. Decidualization and uNK cell activation are initiated by implantation, thus, uNK cell deficits do not influence mouse embryo implantation. 17,22 UNK cells proliferate rapidly within decidua 2,3 but recently it was established that self renewing uNK progenitor cells do not reside there. Uterine segments from normal mice were grafted by end to end anastomosis into uNK cell deficient or normal recipients who were then mated. uNK cells were generated only when hosts had NK cell progenitors. 33,34 In pregnant mammals, thymus and marrow involute during pregnancy 35,36 while spleen and lymph nodes (LN) become hypercellular.
  • LN lymph nodes
  • uNK cells appear to be recruited to decidua basalis and then move into the mesometrial triangle, the entry portal for nerves and vessels supplying the uterus and developing feto-placenta units. 3,39,40 In decidua basalis of normal mice at mid gestation, 7% of uNK cells are within vessels homologous to spiral arteries, another 30% are in walls of these vessels and the remaining cells, as resolved in paraffin- embedded sections, are associated with other tissues. 41
  • uNK cells are a major source of inducible nitric oxide synthase, 22 an IFN- ⁇ regulated enzyme producing the powerful vasodilator nitric oxide (NO).
  • NO vasodilator nitric oxide
  • uNK cell deficient mice expression of this enzyme is induced in trophoblast but at very low levels 22 that cannot dilate the spiral arteries (Kiso and Croy, unpublished vascular casting data). Ineffective dilation of spiral arteries is a hallmark of the human gestational complication hypertension/pre-eclampsia. 42,43 Despite extensive study of this syndrome, its frequency remains constant and there is no consensus on underlying causes. 44,45 Systemic endothelial cell damage underlies clinical symptoms and may be mediated by dysregulated blood cytokine balance.
  • Lymphocytes constitutively express the tethering molecule L-selectin, which interacts with Peripheral LN Addressin (PNAd) and Mucosal Vascular Addressin-1 (MAdCAM-1) expressed by the microvillous surface of endothelium in LN and Peyers Patches (PP). Avidity of these interactions is modulated by physiological responses including cytokines, inflammation and fever which trigger rolling for egress of non activated lymphocytes from vessels. 5,66"68 Firmer adhesion and transendothelial migration involve integrins, particularly ⁇ 4 ⁇ 7 which uses MAdCAM-1.
  • VCAM-1 Vascular Cell Adhesion Molecule 1
  • LFA-1 Leukocyte Function Associated Antigen-1
  • NK cells are required to maintain the integrity of the decidualized uterine stroma and for initiating decidual artery instability. Failure to recruit adequate uterine NK cells to the human uterus may lead to a collapsing decidua that would limit implantation success or contribute to gestational hypertension with or without preeclampsia.
  • the inventors have determined that uterine NK cells do not self- renew in the uterus but rather are recruited to the uterus from the periphery. It is expected that cyclic endocrine changes in the late menstrual cycle and early pregnancy in women, open molecular gates in uterine endothelium that promote movement of NK cells and their precursors into the uterus. Further, the inventors postulate that defects in appropriate NK cell trafficking compromise establishment of pregnancy and/or lead to patients being classified as infertile or pre-eclamptic.
  • the present invention provides a method of monitoring the luteal phase of a menstrual cycle and/or a pregnancy in a female by observing the adhesion of lymphocytes from the female with uterine or lymphoid tissue from a pregnant animal.
  • the invention also includes the identification of lymphocyte subsets involved in the adhesion and to the determination of the effect of ovarian hormones on the interactions between lymphocytes and uterine endothelium.
  • the invention further relates to the determination of the effect of controlled ovarian hyperstimulation (COH) with fertility drugs on the interaction between the lymphocytes and the uterine endothelium.
  • COH controlled ovarian hyperstimulation
  • the present invention further relates to kits for performing the method of the invention.
  • Figure 1 shows photomicrographs of grafted uterine segments at day 10 of gestation. Boxed regions in A, C, E are enlarged in B, D and F.
  • A&B Lower and higher power images of a CD1 graft site in a CD1 recipient containing a normally developed implantation site including the mesometrial aggregation of lymphocytes (MLAp), decidua basalis (DB) and placenta (pi).
  • MLAp mesometrial aggregation of lymphocytes
  • DB decidua basalis
  • pi placenta
  • B shows numerous, mature uNK cells (open arrowheads).
  • C&D Lower and higher power images of a SCID graft segment in a tg ⁇ e26 recipient revealing development of a uterine stroma- derived deciduoma (DC).
  • DC deciduoma
  • Image D is representative of the serial sections of the deciduomatae which contained no uNK cells.
  • E UNK (arrowheads) cells established within the decidual basalis by spleen cells from gestation day (gd 5) donors were mature granulated cells.
  • F Spleen cells from non- pregnant donors did not generate uNK cells in the 10 day assay protocol.
  • the blood vessel (asterisk) is an unmodified decidual spiral artery.
  • MT mesometrial triangle, DB, decidua basalis, pi placenta.
  • A-F stained with PAS. Bars in A&C 400 ⁇ mm; in B, 40 ⁇ mm and in D-F, 25 ⁇ mm.
  • Figure 2 shows the representative photomicrographs (160x) comparing adhesion of lymphocytes from a single donor to mouse LN from virgin or d ⁇ pregnant B6 mice with and without anti L-selectin (DREG-56) blocking.
  • B Summary of results from a single donor across the mouse gestational time course. Functional adhesive changes occur during early decidualization.
  • C Large and small CD56 b ⁇ ght cell adhesion to non- pregnant uterus or Day 10 decidua basalis (x200).
  • D Summary of results from a single donor whose cells were pre labelled with CD56 and a PE- conjugated secondary antibody.
  • Figures 3A and 3B are bar graphs showing that pregnancy stimulates lymphocyte adhesion in uterine and peripheral lymph node tissues.
  • Uterine or peripheral lymph node (PLN) tissues were isolated from non-pregnant (NP) mice or pregnant mice at various times of gestation, cryopreserved and used in adhesion assays performed under shear. Prior to assay, peripheral blood lymphocytes (PBL) were incubated for 30 minutes with the indicated function-inhibiting mAb specific for L-selectin (DREG-56) or alpha4 integrin (HP2/1). Data are the mean + SD of triplicate samples; results are representative of 3 independent experiments. The differences between adhesion of CD56 bright cells or unlabeled PBL to tissues from NP or pregnant mice were determined to be significant, p ⁇ 0.0001 (*), using an unpaired two-tailed Student's t test.
  • Figure 4 is a bar graph showing the effect of pregnancy on the L- selectin-specific adhesion of murine splenocytes to mouse lymph node endothelium. Assays on lymph node substrate tissue from nonpregnant mice showed increased adhesive interactions when the splenocytes came from a pregnant compared to nonpregnant donor. Further, the splenocytes from mid-pregnancy (gd 10 were more adhesive than those from the first trimester (gd 6)
  • Figure 5 provides bar graphs showing the effect of estrogen treatment on (A) human blood lymphocyte adhesion to lymph node endothelium, where the ovariectomized endothelium donor was treated or untreated with estrogen (panel LN HEV); (B) TK1 indicator cell line (L- selection negative, alpha4 integrin high) adhesion to Peyer's Patches endothelium, where the endothelium donor was treated or untreated with estrogen (panel PP HEV); (C) human blood lymphocyte adhesion to mouse pancreatic endothelium, where the endothelium donor was treated or untreated with estrogen (panel Pancreas); and (D) murine splenocyte adhesium to nonpregnant uterine endothelium, where the splenocyte donor was treated or untreated with estrogen (panel Splenocyte).
  • A human blood lymphocyte adhesion to lymph node endothelium, where the ovariectomized endothelium donor
  • Figure 6 is a bar graph comparing the effect of estrogen to natural pregnancy on the ability of uterus to preferentially bind CD56 ⁇ ght NK cells. Effects were similar.
  • Figure 7 is a bar graph showing the effect of progesterone on the adhesion of human blood lymphocytes to murine lymph node endothelium as a substrate (indicator tissue from LN of a non-pregnant mouse).
  • Progesterone alone is equal to estrogen alone and either promotes adhesion that is elevated above that seen in tissues from virgin intact females or in ovariectomized females given oil as the appropriate placebo treatment.
  • Figure 8 is a bar graph showing the effect of progesterone on the adhesion of human blood lymphocytes to murine Peyer's Patches endothelium as a substrate.
  • Progesterone alone is equal to estrogen alone and either promotes adhesion that is elevated above that seen in tissues from virgin intact females or in ovariectomized females given oil as the appropriate placebo treatment.
  • Figure 9 is a bar graph showing the effect of progesterone on the adhesion of murine splenocytes to murine endothelium as a substrate (splenocyte donor treated or untreated with hormone).
  • Progesterone alone is equal to estrogen alone and either promotes adhesion that is elevated above that seen in tissues from virgin intact females or in ovariectomized females given oil as the appropriate placebo treatment.
  • Figure 10 is a bar graph showing the effect of progesterone on the adhesion of CD56 br ⁇ ght NK cells to murine uterine tissues.
  • Progesterone alone is equal to estrogen alone and either promotes adhesion that is elevated above that seen in tissues from virgin intact females or in ovariectomized females given oil as the appropriate placebo treatment.
  • Figure 11 is a schematic showing the inoculation protocols for Example 4.
  • Figure 12 is a bar graph showing the mean numbers (+ standard deviations) of human CD56 b ⁇ ght Natural Killer cells bound to uterine stroma from ovariectomized mice treated with oil, low dose 17- ⁇ estradiol (100ng/day, E2lo), progesterone (1mg/day, P4), combined E2 plus P4 (E2/P4) or combined E2 and P4 (E2/P4) plus induction of uterine decidua (E2/P4+deciduoma).
  • Figure 12 is a bar graph showing mean numbers ( ⁇ standard deviations) of human lymphocytes bound to high endothelial venules (HEV) in peripheral lymph nodes (PLN) from ovariectomized mice treated with oil placebo, low dose 17- ⁇ estradiol (100ng/day, E2low), progesterone (1 mg/ day, P4), combined E2 plus P4 (E2/P4), or combined E2 and P4 (E2/P4) plus induction of uterine decidua (E2/P4+deciduoma).
  • HEV high endothelial venules
  • Figure 13 is a bar graph showing mean numbers ( ⁇ standard deviations) of TK-1 cells, a mouse lymphoma cell line expressing 4 ⁇ 7 integrin as its only known endothelial adhesion receptor, on high endothelial venules (HEV) in Peyer's Patches (PP) from mice treated with low dose 17- ⁇ estradiol (100ng/day, E2low), progesterone (1mg/ day, P4), combined E2 and P4 (E2/P4), or combined E2 and P4 (E2/P4) plus decidual induction (E2/P4+deciduoma).
  • HEV high endothelial venules
  • Figure 14 is a bar graph showing mean numbers ( ⁇ standard deviations) of mouse splenocytes bound to high endothelial venules (HEV) of peripheral lymph nodes (PLN) from non-pregnant virgin Balb/c mice.
  • HEV high endothelial venules
  • PPN peripheral lymph nodes
  • pre-uNK immediate precursors of uNK cells self-renew within uterus or are recruited from the periphery.
  • uterine segments from NK cell competent mice were grafted orthotopically into NK/uNK cell deficient or wildtype mice. Recipients were mated and graft sites were examined histologically. Only decidualized grafts in wildtype recipients contained uNK cells, indicating uterus does not self-renew pre- uNK cells.
  • thymus, bone marrow (BM), lymph node (LN) or spleen was grafted from virgin or pregnant NK cell competent donors into mated NK/uNK cell deficient recipients. Only secondary lymphoid tissue gave high levels of uNK cell reconstitution. Reconstitution by splenocytes required donor pregnancy but was independent of precursor expression of the chemokine receptors CCR2 or CCR5. Lymphocyte recruitment into tissues requires adhesive interactions with vascular endothelium. This was assessed by examining adhesion of human lymphocytes to frozen mouse tissue sections under shear.
  • the inventors conducted experiments to assess involvement of lymph nodes (LN) in trafficking/recruitment of uterine Natural Killer (uNK) cells during mouse gestation. 693
  • the Stamper-Woodruff assay of cell adhesion to frozen tissue sections under shear takes advantage of the fact that human lymphocytes bind to murine adhesion molecules 70 and was used to determine if pregnancy in C57BI/6 mice modified adhesion of lymphocytes to lymph node endothelium.
  • Human lymphocytes were prepared as indicator cells from single blood donor buffy coats purchased from the American Red Cross. Consistent results have been obtained with 20 donors although no information is available regarding sex or possible menstrual cycle stage of the donors.
  • lymphocyte preparations were assessed for adhesion to murine Peyer's Patches, pancreas and uterine tissue sections. Dynamic changes similar to those in Lymph Nodes were found in Peyer's Patches. Importantly, no pregnancy-associated changes occurred in pancreas, showing pregnancy regulates leukocyte endothelial interactions in selected tissue micro-environments. Adhesion was infrequent to nonpregnant uterus, (between endometrium and myometrium mesometrially and antimesometrially) but high numbers of human cells localized to decidua basalis of pregnant uteri.
  • mice were 1) normal cycling virgin young adult females; 2) naturally mated, gestationally timed primiparous females; 3) ovariectomized virgin adult females treated with a placebo; 4) ovariectomized virgin adult females receiving estrogen replacement therapy; 5) ovariectomized virgin adult females receiving progesterone replacement therapy; 6) ovariectomized virgin adult females receiving combined estrogen plus progesterone replacement therapy; and 7) ovariectomized virgin adult females receiving combined estrogen plus progesterone replacement therapy plus induction of uterine decidua.
  • lymph nodes from only nonpregnant mice as a substrate, it was shown that splenocytes collected from pregnant mice were more adhesive to nonpregnant lymph node endothelium than the splenocytes isolated from non pregnant mice (Figure 4). Further, the lymphocytes from mid pregnancy (gd 10) were more adhesive than those from the first trimester (gd 6).
  • PNAd Peripheral Node Addressin
  • the elevated adhesion to blood vessel endothelium (a requisite for moving a cell from the circulation and into a tissue) is a combined result of the effect of pregnancy on both organ specific endothelium and on the lymphocytes themselves. This interaction was also shown to involve alpha4 integrin-mediated pathways.
  • TK1 human blood lymphocytes
  • endothelium was not universally altered in the estrogen-treated females as there was no increase in adhesion of blood lymphocytes, or of the cell line that only has alpha4 integrin adhesiun receptors, to pancreas endothelium.
  • Estrogen replacement also had a major effect on lymphocytes themselves, directly promoting their adhesion to endothelium from nonpregnant donors.
  • the adhesion pathway was shown to involve the L-selectin molecule since an antibody specific to L-selectin could block the functional interaction between the splenic lymphocytes and the lymph node endothelium.
  • Estrogen also altered endothelial cell-lymphocyte interactions in assays using uterine tissues.
  • the percentage of CD56 b ⁇ ght cells in the starting human lymphocyte preparation was 2-3%.
  • the percentage of CD56 br ⁇ ght cells was greatly enriched (2.5 fold) to 10%.
  • estrogen induced changes in uterine endothelium were proven to promote interactions specifically with human CD56 br ⁇ ght cells, the cell subset that normally homes to the uterus in pregnancy.
  • progesterone independent of estrogen, can modify lymphocyte-endothelial cell interactions.
  • human blood lymphocytes were shown to have enhanced, L-selectin dependent binding (above that measured in tissues from non-pregnant or ovariectomized placebo treated females) to peripheral lymph node endothelium from a mouse receiving progesterone replacement therapy.
  • the alpha4 integrin-expressing TK1 cell line was shown to have enhanced, progesterone-dependent, and alpha4 integrin- dependent, binding to endothelium in Peyer's Patches.
  • Progesteone also acts on lymphocytes themselves (murine) to promote binding to endothelium from a single source, lymph nodes from non pregnant females. Adhesion of human CD56 bnght cells to uterine gestational tissues was also promoted in ovariectomized, progesterone treated mice. The dual treatment of the ovariectomized mice with estrogen and progesterone did not enhance the interactions of human CD56 bnght cells with uterine stroma from the treated mice beyond those seen with treatment using only single steroid therapy.
  • mice receiving dual treatment with estrogen and progesterone as well as induction of uterine decidua indicate that the enhanced interactions between human CD56 b ⁇ ght cells and uterine stroma from the treated mice in the presence of estrogen and/or progesterone occur independently of whether or not the uterus has undergone decidualization of its stromal cells.
  • CD56 b ⁇ ght human blood lymphocytes (the human uterine phenotype) are enriched when interacting with uterine endothelial cells from non pregnant uteri and very strongly enriched when interacting with uterine endothelial cells from pregnant uteri.
  • a technical assay for studying the interaction between lymphocytes and endothelium from uterine and/or lymph nodes is valid for many blood donors and that the assay gives consistent results when applied to studies of gestational lymphocytes and endothelium.
  • the present invention provides a method of monitoring the luteal phase of a menstrual cycle and/or a pregnancy in a female by detecting or assessing the adhesion of lymphocytes from the female with uterine or lymphoid tissue from a pregnant animal or from a non-pregnant animal that has been treated with steroids such as estrogen and/or progesterone.
  • the method can be used to assess the luteal phase of a woman trying to conceive to determine if the uterine environment is conducive for sustaining a pregnancy.
  • the method will have particular utility in women having trouble conceiving, women experiencing habitual miscarriages and women undergoing in-vitro fertilization (IVF).
  • the assay of the invention can be used to determine if the problems in conceiving or maintaining a pregnancy are related to problems in lymphocyte adhesion.
  • the method of the invention can also be used to monitor an early pregnancy wherein the greater the adhesion the greater the chance of sustaining the pregnancy.
  • the method or assay of the invention comprises:
  • the female can be any female animal wherein one desires to monitor the luteal phase or pregnancy.
  • the female is preferably a human female.
  • the lymphocytes can be obtained from any sample from the female and are preferably obtained from blood or fractions thereof.
  • the lymphocytes used in the assay are preferably peripheral blood leukocytes that may be tagged to identify the CD56 b ⁇ ght natural killer cell subset. Most preferably, the lymphocytes are CD56 br ⁇ ght natural killer cells.
  • the term "uterine or lymphoid tissue” includes sections or homogenates of the tissue that may be useful in the method.
  • the uterine or lymphoid tissue can be from any animal and are preferably from a mouse, rat, golden hamster, guinea pig, rabbit, humans or other species in which decidual tissues develops in the pregnant or pseudopregnant uterus.
  • the uterine tissue is preferably from the decidua basalis.
  • the lymphoid tissue can be from any lymphoid tissue and is preferably from the lymph node or Peyer's Patches. In the assay histological sections or homogenates of the tissue may be used.
  • the tissue or homogenates may be placed on or adhered to a coverslip or microtiter plate to which the lymphocytes can be directly applied.
  • greater adhesion of the lymphocytes indicates a better uterine environment for sustaining a pregnancy.
  • the term "greater adhesion of lymphocytes” means the adhesion of lymphocytes from the female is greater with a pregnant animal or a non-pregnant animal that has been treated with estrogen and/or progesterone than with a control.
  • a suitable control may be, for example, uterine or lymphoid tissues from a non-steroid treated, non-pregnant animal or a suitable non-lymphoid or non-uterine tissue, for example, endothelium from the pancreas.
  • lymphocytes may be enumerated by microscopic observation as described in Example 1 , either by staining with, for example, toluidine blue, or using a fluorecent label. Automated scoring based on differential spectroscopy or colorimetric measurement of stained lymphocytes may also be used.
  • the invention also includes the identification of lymphocyte subsets involved in the adhesion and to the determination of the effect of ovarian hormones on the interactions between lymphocytes and uterine endothelium.
  • the invention further relates to the determination of the effect of controlled ovarian hyperstimulation (COH) with fertility drugs on the interaction between the lymphocytes and the uterine endothelium.
  • COH controlled ovarian hyperstimulation
  • kits for use in monitoring the luteal phase of a menstrual cycle and/or a pregnancy of a female The kits would comprise the reagents suitable for carrying out the methods of the invention, packaged into suitable containers and providing the necessary instructions for use.
  • the present invention includes a kit for monitoring the luteal phase of a menstrual cycle and/or a pregnancy of a female comprising uterine or lymphoid tissue from a pregnant animal or a nonpregnant animal that has been treated with estrogen and/or progesterone.
  • the tissues are mounted on a solid support.
  • tissues may be adhered to a coverslip or a microtitre plate.
  • kits may also include reagents to separate lymphocytes from blood and/or reagents, for example antibodies, for tagging or separating the desired lymphocyte subset, for example CD56 br ⁇ ght natural killer cells, from the blood or the lymphocytes.
  • reagents to separate lymphocytes from blood and/or reagents for example antibodies, for tagging or separating the desired lymphocyte subset, for example CD56 br ⁇ ght natural killer cells, from the blood or the lymphocytes.
  • kits With particular regard to assay systems packaged in "kit” form, it is preferred that assay components be packaged in separate containers, with each container including a sufficient quantity of reagent for at least one assay to be conducted.
  • a preferred kit is typically provided as an enclosure (package) comprising one or more containers for the within-described reagents.
  • the reagents as described herein may be provided in solution, as a liquid dispersion or as a substantially dry powder, e.g., in lyophilized form.
  • the reagents are packaged under an inert atmosphere.
  • Printed instructions providing guidance in the use of the packaged reagent(s) may also be included, in various preferred embodiments.
  • the term "instructions" or “instructions for use” typically includes a tangible expression describing the reagent concentration or at least one assay method parameter, such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions, and the like.
  • Example 1 Goals of Example 1 were i) to determine if uterus self renews uNK cells ii) to examine peripheral lymphoid tissues as sources for pre-uNK cells and iii) to explore underlying chemokine and adhesion-related mechanisms that could contribute to mobilization of peripheral cells into decidualizing uterus.
  • MATERIALS AND METHODS Animals:
  • mice Immunocompetent mice (randombred CD1 , Charles River Laboratories, St. Constant, PQ), C57BI/6J (B6) and C57BI/6x129/J F1 (F1, Jackson Laboratories, Bar Harbor, ME)) were housed under standard husbandry in the Central Animal Facility, University of Guelph.
  • mice (randombred ICR- scid/scid (SCID; NK+,T-,B-, Taconic, Germantown, N.Y), tg ⁇ e26 (H- 2k/b ; NK “ ,T “ ,B + ) and RAG ⁇ T ⁇ c " ' " (H-2 b ; NK “ ,T “ ,B “ ) were housed in the University of Guelph's barrier-husbandry facility. Both tg ⁇ e26 or RAG ⁇ ' / ⁇ c " " lack uNK cells and are referred to as uNK cell deficient.
  • CD1 females were anaesthetized (0.35 ml of xylaxine (20mg/ml) and ketamine (100 mg/ml)) and the donor horn was reanastomosed with simple interrupted 8-0 Vicryl (Polysorb®, Norwalk, CT) sutures.
  • donors CD1 or SCID
  • recipients tg ⁇ e26 or CD1 were anaesthetized as above.
  • Thymic engraftment Thymuses were dissected from non-pregnant or pregnant (gd 3, 5) adult or neonatal (48 hr) B6 mice and grafted under the renal capsule of anaesthetized gd 0 RAG-2 "/ 7 ⁇ c " " . Adoptive transfer of BM, LN or splenocytes.
  • Bone marrow (BM) and spleen cell (SC) donors were non-pregnant or pregnant SCID mice while lymph node (LN) donors were B6 (gd 3, 5 or 7). As pregnancy changes cellularity of these organs, one donor was used per recipient, pooling donors if several mated recipients were available on the same day. Uteri from all gd 3 donors were flushed to confirm pregnancy by detection of pre-implantation blastocysts. BM was flushed from femurs and tibias of each donor. Microscope-aided dissection was used to harvest peripheral (P) LNs (9 superficial and 3 pelvic LN/donor) that were pooled and the mesenteric (M) LN chain.
  • P peripheral
  • M mesenteric
  • PBS 400 ⁇ ml with/without cells
  • gd 0 tg ⁇ e26 or RAG-2-/-/ ⁇ c-/- recipients who were sacrificed on their gd 10.
  • Circular smooth muscle was used as the boundary between these.
  • mesometrial triangle (MT) rather than MLAp is the term used to describe the scored region.
  • MT mesometrial triangle
  • Means and standard deviations of uNK cells/mm 2 , p values and Student-Newman-Keul test for ANOVA were conducted using PC-SAS 6.12 for Windows (SAS Institute Inc., NC). Lymphocyte adhesion to frozen mouse tissues under shear.
  • PBL Human peripheral blood lymphocytes
  • nonpregnant donor buffy coat leukocyte concentrates American Red Cross, Buffalo, NY by Ficoll/Hypaque centrifugation and adherent cell depletion.
  • PBL peripheral lymph node
  • 68 Uteri and (peripheral lymph node (PLN) tissues from non-pregnant and pregnant (gd 3,6,10) B6 mice were cryopreserved immediately upon collection.
  • PBL were unstained or stained with anti-CD56 mAb or isotype- control lgG1 (Coulter Immunology, Hialeah, FL) and goat anti-mouse IgG- RITC Ab to permit analysis of NK cell subset adhesion, as previously described.
  • PBL Prior to assay, PBL were incubated with or without blocking mAb (10ug/ml) specific for L-selectin (DREG-56, American Tissue Type Culture Collection, Rockville, MD) or alpha4 integrin (HP2/1 , Coulter).
  • mAb 10ug/ml
  • L-selectin DREG-56, American Tissue Type Culture Collection, Rockville, MD
  • alpha4 integrin HP2/1 , Coulter
  • uNK cell frequency is 27- 53 cells/mm 2 in DB and 72-129 cells/mm 2 in MLAp (Table 2).
  • UNK cell sizes in B6 mice ranged from 11-20 ⁇ m (average 14.3 ⁇ 2.9) and in SCID mice 11-27 ⁇ m (mean 15.9 ⁇ 4.0). All uNK cells were granulated and contained 8-35 (mean 18.0+8.2) and 5-30 granules/cells (mean 19.4 ⁇ 9.0) in B6 and SCID mice, respectively.
  • Uterine segment transplantation suggested that migration of pre-uNK cells accounts for filling of these microdomains.
  • Peripheral lymphoid tissues were assessed for pre-uNK cells by grafting to mated, uNK cell deficient mice. Thymic engraftment generated limited numbers of uNK cells at gd 10 (Table 2). There were no statistical differences in reconstitution of DB or MT by thymuses of different ages or from different donor pregnancy states (p>0.05). BM from nonpregnant or 3 early times of pregnancy also gave low level uNK cell reconstitution in all recipients (Table 2). No significant differences were found in uNK cells/mm 2 in DB or MT between the BM donor groups (p>0.05). MLN failed to reconstitute uNK cells while implantation sites in recipients of PLN showed MLAp development.
  • Both MLAp and DB of PLN grafted mice contained mature uNK cells. Implantation sites in uNK cell deficient mice receiving SC from pregnant donors also showed histological development of MLAp and high levels of uNK cells in both MLAp and DB. However, if the SC donors were not pregnant, uNK cell reconstitution was much lower (p ⁇ 0.001) in both microenvironments. As shown in Fig. 1(E&F), levels of engraftment resulting from inoculation of SC from pregnant donors was sufficient to modify the decidual spiral arteries. In sharp contrast, host arterial vasculopathy persisted in recipients of SC from non-pregnant donors.
  • uNK cells were present at higher frequencies in the MLAp than in DB (p ⁇ 0.01), a typical gd 10 pattern in normal mice.
  • Morphological assessment of graft-derived uNK cells showed that uNK cells derived from thymus, BM, LN and SC were similar in size (14.5+4.0, 15.6+4.5, 18.7 ⁇ 7.5 and 13.9 ⁇ 4.7 ⁇ m, respectively) and in numbers of granule/cell (12.8+5.8, 15.6 ⁇ 5.5, 20.5 ⁇ 11.9, 17.8 ⁇ 7.7, respectively).
  • These morphology were identical to gd 10 uNK cells in unmanipulated B6 and SCID mice, implying equivalent maturity.
  • Human anti-CD56 mAb-labeled cells were assessed for adhesion under shear to frozen uterine sections from non-pregnant or pregnant B6 mice (gd 3, 6, 10). The proportion of labeled cells in test suspensions was -10-12% CD56 bright with ⁇ 1.5% being CD56 bright .
  • Pre-Iabeling with anti- CD56 did not affect the total number of cells bound to uterine tissues at any of the time points (data not shown).
  • CD56 br ⁇ ghf cells adhered to non-pregnant endometrial stroma in a randomly-dispersed, low frequency manner (Fig. 3). Numbers of cells adhering to to uterine stroma increased in gd 3 tissue but remained randomly distributed.
  • Pregnancy-induced adhesion was dependent on both the L-selectin and alpha4 integrin homing receptors (Fig. 3) since it was inhibited by specific function blocking antibodies (i.e. DREG-56 and HP2/1, respectively) but not by an lgG1 isotype-matched control antibody.
  • Pregnancy induced dramatic and unexpected gains in lymphocyte adhesion in HEV of PLN from gd 3,6,10 B6 mice (Fig. 3).
  • lymphocyte adhesion to HEV from non-pregnant and pregnant mice was L-selectin- dependent and did not involve alpha4 integrin.
  • 73 Only normally-sized lymphocytes bound to HEV.
  • mice This is the first comprehensive study, in any species, to address the source of the immediate precursors of uNK cells in a pregnant adult. Availability of NK/uNK cell deficient mice that reliably carried pregnancies was central to the study's success. Following transplantation of uterine segments from NK + mice into NKVuNK " mice, no uNK cells were found in decidual tissue within the grafts or at any of the implantation sites in host tissue. The latter observation excluded migration of pro/pre-uNK cells from the graft segments into host tissue and established that mouse uterus does not contain self-renewing pro/pre-uNK cells.
  • Uterine and oviductal grafts used in published studies, may have scarred and died due to problems of excessive length, inadequate perfusion and/or immune rejection.
  • the choice of an immune deficient host eliminated host versus graft rejection.
  • Early graft versus host disease was not a problem as allografts from CD1 were as equally viable and hormone responsive as T cell deficient SCID allografts.
  • Duration of the transplantation experiments was shorter (17 days) than mouse gestation (19-20 days), permitting the conclusion that uterine recruitment likely occurs during gestations.
  • Previous grafting of mated, immunocompetent mice with virgin uterine tissue in sealed diffusion chambers showed that uterus has some pre-uNK cells with a 12 day maximum survival time. 74
  • the developmental stages of hematopoietic cells which move into the uterus are not yet known. Because uNK cells differentiating from thymus, BM, LN and SC are identical morphologically and morphometrically and match those in gd 10 unmanipulated, genetically normal mice, the cells which moved into the uterus from these tissues were probably at relatively similar stages of differentiation.
  • uNK cells may differentiate rapidly and cells at various pro/pre-uNK stages may have had sufficient time to complete differentiation under the experimental conditions described herein.
  • the heterogeneity in size of human lymphocytes adhering to murine uterus suggests that circulating cells at more than one stage of differentiation/activation may have uterine homing potential.
  • Lack of CCR2 or CCR5 did not reduce pre-NK cell homing from spleen to uterus despite high levels of CC chemokine expression in pregnant human and mouse uteri 79,80 suggesting that these chemokines target other cell types, and/or that there is a redundancy in uterine chemokines adequate to recruit pre-uNK cells through other receptors.
  • Adhering cells were absent over fetal trophoblasts and maternal MT/MLAp regions but localized between these microenvironment, exclusively over DB. In gd 10 mice, about 10% of the large, mature uNK cell population is intravascular in DB while intravascular uNK cells are rare in MLAp, a domain containing uNK cells with less mature morphology. 81 Thus, adherence of CD56 bnght cells in DB could indicate an endothelium participating in pre-uNK cell recruitment and/or endothelial targets of uNK cell functions within implantation sites. Adhesion was dependent on L-selectin and alpha4 integrins. Coordinate expression of these molecules has been previously documented on circulating CD56 b ⁇ ght cells.
  • L-selectin and alpha4 integrins function as gatekeepers controlling lymphocyte extravasation through their ability to bind to vascular addressins under shear. 73,82 It is plausible to speculate that pregnancy induces differential display of vascular adhesion molecules on uterine microvessels. Recent studies showed VCAM-1 is restricted to endothelium of the central DB. 40 While this could account for the alpha4 integrin- mediated binding we observed, caution must be used in this interpretation since extracellular matrix is also exposed in the assayed tissue and alpha4 integrins bind to fibronectin, a molecule prevalent in mesometrial decidua.
  • L-selectin's ligand is undefined. Kruse et al. reported MAdCAM-1 expression on mesometrial endothelium in the vascular zone, lateral to the DB 40 MadCAM-1 is a counter-receptor for L-selectin and 4 ⁇ 7 in Peyer's Patches and may have this function in decidualized uterus.
  • an L-selectin ligand, 84 is upregulated mesometrially between gd 6 and 10 in B6 uterus while PSGL-1 , a P-selectin ligand 85 used by NK cells is constantly expressed at these times (unpublished microarray data). Regardless of the mechanisms involved, exposure of PBL to pregnant uterine tissue results in preferential adhesion and enrichment of CD56 br ⁇ ght cells (Fig. 3).
  • mice and tissue dissections Female and stud male C57BL/6J mice, aged 7-8 weeks, were purchased from the Jackson Laboratory, Bar Harbor, ME, and housed and mated under conventional husbandry at the University of Guelph. The day a copulation plug was detected was called day 0 of gestation (gd 0). Females were exported to Buffalo and killed at specific timed intervals from the day of the copulation plug. Non-pregnant controls were always unmated, virgin females.
  • Euthanasia was by cervical dislocation and all procedures were covered by appropriate animal utilization protocols at both institutions. Up to five tissues were recovered/mouse by dissection, placed immediately into OCT (Miles Laboratories), frozen in liquid nitrogen-cooled isopentane and stored at -20°C for as long as 10 days before crystat sectioning. Any tissue block was used only once or twice before it was discarded as potentially degenerating.
  • the dissected tissues were I) a pool of approximately 10 peripheral lymph nodes from subcutaneous or intermuscular sites; ii) a pool of the para-aortic lymph nodes that drain the uterus; iii) a pool of 10-12 intestinal Peyer's Patches (PP); iv) the entire pancreas and v) the uterus.
  • each horn was incised longitudinally in the anti- mesometrial quadrant.
  • Cryostat cutting occurred from the mucosal surface along the length of each horn.
  • the horns were incised between each implantation site and, with very sharp Vannas scissors or a fresh surgical blade, the implant site was incised in the medial plane.
  • Cryostat cutting was parallel to the cut surface, to represent the center of each implantation site. Three replicate tissue sections were mounted/coverslip for each specimen.
  • Example 2 Effect of pregnancy on lymphocyte adhesion to endothelium Using lymph nodes from only non-pregnant mice, it was shown that lymphocytes collected from pregnant donors were more adhesive to endothelium than the lymphocytes isolated from non pregnant mice ( Figure 4). Antibody blocking of the Peripheral Node Addressin (PNAd) receptor reduced adhesion, confirming involvement of the ligand for this receptor, L- selectin as a key molecule involved in the process. Thus, the elevated adhesion to blood vessel endothelium (a requisite for moving a cell from the circulation and into a tissue) is a combined result of the effect of pregnancy on both organ specific endothelium and on lymphocytes themselves. This interaction was shown to involve at least two adhesion pathways, the L- selectin-mediated ( Figure 4) and alpha4 integrin-mediated pathways (not shown).
  • Example 3 Example 3:
  • tissue sections were prepared from ovariectomized mice treated with placebo (oil, the solvent for the hormone preparations) or estrogen alone or progesterone alone.
  • placebo the solvent for the hormone preparations
  • the positive control tissues in the experiment came from naturally mated mice and the negative control tissues were from non pregnant mice.
  • Figure 5 shows a representative data outcome from the experiment involving estrogen replacement.
  • estrogen was equivalent to pregnancy in promoting lymphocyte adhesion to endothelium under shear flow conditions (see panel LN HEV). This functional interaction was again blocked by function-blocking antibodies for the L-selectin receptor.
  • the cell line TK1 was used instead of human blood lymphcytes. This cell line lacks adhesion receptors for L- selectin but highly expresses alpha4 integrin.
  • the data in this panel show that estrogen induces adhesion of cells that bind via alpha4 molecules.
  • the blocking antibody in this panel confirms this conclusion.
  • the top row of Figure 5 shows that estrogen replacement therapy acts on at least 2 adhesion pathways (L-selectin-mediated and alpha-4 integrin-mediated) to alter endothelium to promote adhesion of lymphocytes.
  • Panel "Pancreas” shows that endothelium is not universally altered in the estrogen-treated females because there is no increase in adhesion of blood lymphocytes or of the transfected cell line.
  • Estrogen replacement also has a major effect on lymphocytes themselves, directly promoting their adhesion to endothelium. This is shown in the panel labeled "Splenocytes" in Figure 5.
  • the endothelium was kept constant and was from a non pregnant donor.
  • the splenocytes were isolated from normal cycling virgin young adult female mice, ovariectomized virgin adult female mice treated with a placebo or ovariectomized virgin adult female mice receiving estrogen replacement therapy. Elevated adhesion was not seen in the placebo treated group.
  • the adhesion pathway that had been measured was shown to involve the L- selectin molecule since an antibody specific to L-selectin could block the functional interaction between the splenic lymphocytes and the lymph node endothelium.
  • Estrogen also altered endothelial cell-lymphocyte interactions in assays of uterine tissues (Figure 6).
  • the percentage of CD56 bnght cells in the starting human lymphocyte preparation was 2-3%.
  • the percentage of CD56 b ⁇ ght cells was greatly enriched 2.5 fold to 10%.
  • There was further enrichment to 70% is the uterine tissue was from a mouse receiving hormone therapy and this enrichment was not different to that seen for a pregnant uterus.
  • estrogen induced changes in uterine endothelium were proven to promote interactions specifically with human CD56 br ⁇ ght cells, the cell subset that normally homes to the uterus in pregnancy.
  • FIGs 7 and 8 show the actions of progesterone on endothelium in lymphoid organs.
  • human PBL were shown to have enhanced, L-selectin dependent binding (above that measured in tissues from non pregnant or ovariectomized placebo treated females) to peripheral lymph node endothelium.
  • the alpha4 integrin expressing TK1 cell line was shown to have enhanced, progesterone- dependent and alpha4 integrin dependent to endothelium in Peyer's Patches.
  • Figure 9 shows that progestrone also acts on lymphocytes themselves (murine) to promote binding to endothelium from a single source, lymph nodes from non pregnant females.
  • Figure 9 data from two independent, ovariectomized, hormonally-treated female mice are shown for each hormone. Adhesion of human CD56 b ⁇ ght cells to uterine gestational tissues was also promoted in ovariectomized, progesterone treated mice ( Figure 10).
  • Example 4 Example 4:
  • mice used as donors of tissues were 6-8 week old young virgin C57BI/6J females who were surgically ovariectomized and then prepared using one of the inoculation protocols illustrated in Figure 11.
  • Killer cells bound to uterine stroma from ovariectomized mice treated with oil, low dose 17- ⁇ estradiol (100ng/day, E2lo), progesterone (1mg/day, P4), combined E2 plus P4 (E2/P4) or combined E2 and P4 (E2/P4) plus induction of uterine decidua (E2/P4+deciduoma) are shown in Figure 12. Numbers of cells bound were counted in 10 high power microscopic fields (5mm 2 each) at 250X magnification. Higher numbers of untreated CD56 br ⁇ ght cells bound to uteri from ovariectomized mice receiving hormones than to uteri from oil-injected control ovariectomized mice (t, p ⁇ 0.05).
  • Mean lymphocytes bound to HEV in PLN from hormone treated animals was significantly higher (t, p ⁇ 0.05) than mean lymphocytes bound to HEV in PLN from oil-injected ovariectomized mice.
  • Treatment of the lymphocytes with DREG-56, a monoclonal antibody (mAb) to human L-selectin significantly blocked human lymphocyte binding to mouse endothelium ( * , p ⁇ 0.05), confirming the identity of the adhesion pathway involved for human lymphocyte binding to HEV in PLN.
  • TK-1 cells Mean numbers ( ⁇ standard deviations) of TK-1 cells, a mouse lymphoma cell line expressing 4 ⁇ 7 integrin as its only known endothelial adhesion receptor, on high endothelial venules (HEV) in Peyer's Patches (PP) from mice treated with low dose 17-b estradiol (100ng/day, E2low), progesterone (1mg/ day, P4), combined E2 and P4 (E2/P4), or combined E2 and P4 (E2/P4) plus decidual induction (E2/P4+deciduoma) are shown in Figure 14. The data were collected from 100 HEV and enumeration was done thrice.
  • TK-1 cells bound to HEV from hormone treated animals were significantly higher (t, p ⁇ 0.05) than cell numbers bound to HEV from of PP from oil-injected ovariectomized mice.
  • Treatment of the TK-1 cells with DATK-32, an monoclonal antibody (mAb) to a4b7 integrin significantly blocked the TK-1 cells binding ( * , p ⁇ 0.05), confirming the mechanism for the adhesion.
  • splencocytes bound to high endothelial venules (HEV) of peripheral lymph nodes (PLN) from non-pregnant virgin Balb/c mice are shown in Figure 15.
  • the splencocytes were obtained from ovariectomized C57BI/6J mice injected with sesame seed oil (oil), low dose 17-b estradiol (100ng/day, E2Io), progesterone (1 mg/ day, P4), combined E2 and P4 (E2/P4), or combined E2 and P4 (E2/P4) plus decidual induction (E2/P4+deciduoma).
  • the data were collected from 100 HEV. Enumeration was done thrice.
  • Mean numbers of bound splenocytes were significantly higher (f, p ⁇ 0.05) when the splenocytes came from hormone-treated ovariectomized mice than from ovariectomized, oil-injected mice.
  • Examples 5-7 aim to define the normal, hormonally- and gestationally-induced changes in adhesion properties of women's lymphocytes with endothelial cells that recruit NK cells into the decidualizing uterus for promotion of successful decidualization, implantation, uterine artery modification and pregnancy.
  • Example 5 requires large numbers of human cells and will involve 8 leukophoresis collections from subjects, 4 at LH+8 and 4 at cycle day 5 (follicular phase). When possible, the same donors will be used for both stages.
  • C57BI/6J mice will be purchased from Jackson Laboratories and maintained for breedings. For Example 5, 20 reproductive stages will be prepared including estrus and postpartum. For Examples 6 and 7, nonpregnant and one gestational time (selected as the plateau adhesion day, likely gd 6) will be used. All pregnancy time points required in a single experiment must be available on the same day. Mice will be euthanised, uterus, LN and pancreas, dissected and placed in OCT compound for immediate freezing in N 2 chilled isopentane and storage. Adhesion molecule stability is 10 days. On assay days, 12 ⁇ m cryostat sections will be cut from selected test tissues and melted onto glass coverslips. Quantitative Frozen Tissue Adhesion Assay under Shear:
  • Lymphocytes will be prepared by Ficoll/Hypaque centrifugation and adherent cell depletioned using established protocols. 5 ' 66,68 5x10 6 cells are routinely used per section; 2x10 6 are adequate. Studies in Example 5 will establish the minimum number of cells that can be used accurately, because limited cell numbers (3x10 7 ) will be available from serially bled patients (Examples 6 and 7) and an optimized protocol for 6-10 tissue sections is required.
  • lymphocytes will be preincubated 30 min at 20°C with Mab (such as DREG-56, an anti-L- selectin function blocking Ab or T51/22, an LFA-1 blocking Ab, both from ATCC, anti-hu alpha4 integrin (Immunotech) anti CD56-PE (PharMingen), anti-CD3-FITC (Becton Dickenson) or isotype matched control antibodies) before being applied to tissue sections in a 100 ⁇ l volume of RPMI medium + 10% bovine serum.
  • Mab such as DREG-56, an anti-L- selectin function blocking Ab or T51/22, an LFA-1 blocking Ab, both from ATCC, anti-hu alpha4 integrin (Immunotech) anti CD56-PE (PharMingen), anti-CD3-FITC (Becton Dickenson) or isotype matched control antibodies
  • tissue sections will be preincubated 30 min at 4°C with antibodies directed against PLNAd (MECA- 79), MAdCAM-1 (MECA-367), VCAM-1 (MK2.7), l-CAM-1 (YN1/1.7) or species appropriate isotype negative control reagents, prior to initiation of adhesion.
  • PLNAd MAdCAM-1
  • MK2.7 VCAM-1
  • l-CAM-1 YN1/1.7
  • species appropriate isotype negative control reagents prior to initiation of adhesion.
  • Antibody dilutions will be determined by prior studies in Dr. Evan's laboratory.
  • adhesion assays are conducted under shear 68,70 and then washed, fixed and stained. 300-500 high endothelial venules are scored 3 times in lymphoid tissues to obtain a mean binding of lymphocytes/HEV.
  • ELISA will be used to quantify estradiol and LH in plasma from leukophoresis donors and patients.
  • lymphocyte/endothelial cell interactions Quantitative lymphocyte tissue adhesion assays under shear will be conducted for 4 leukophoresis donors on LH +8. After 6-12 mo recovery, leukophoresis will be repeated when donors are at cycle day 5. Leukophoresis will provide>10 9 cells which must be prepared and assessed within 48 hr. Lymphocytes will be analysed without antibody treatment, pretreated by appropriate negative controls or blocked with Mab to L-selectin, alpha4 integrin or LFA-1. Mouse tissue sections will be used unblocked or blocked with Mab to PNAd, MAdCAM-1, VCAM-1, and ICAM-1.
  • Lymphocyte aliquots will be prestained with one of CD56, CD16, CD3, or IgM to identify adherent lymphoid subsets or post-stained to assess IFN- ⁇ production.
  • Uterine tissues will be used for the full gestational time course.
  • LN (positive control) and pancreas (negative control) will be used over a more limited time course.
  • Assays will be conducted serially on lymphocytes provided by patient volunteer recipients of fresh embryos following COH until 5 gd 40 pregnancies have been monitored. Adhesion assays will be identical to those in Example 6 and patients will be run concurrently as available. This example will reveal if a standard IVF hormone therapy protocol 87 in any way modifies interactions between lymphocytes and uterine tissue by comparing the results to those from Example 6. The information gained may lead to reassessment of ovulation protocols or to implementation of hormone therapy for other types of patients. It will be of great interest to understand if pregnancy failure in both of the patient groups correlates with recognized functional adhesion patterns that differ from those of women diagnosed as pregnant. The experimental design also permits correlations of changes in adhesive functions to circulating hormone levels.
  • # of fetuses in maternal density of #. of fetuses number graft host tissues in graft uNK cells at segments graft sites*

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Abstract

L'invention concerne des méthodes d'évaluation ou de contrôle de la phase lutéale du cycle menstruel et/ou de la première moitié de la grossesse par l'évaluation de l'adhésion des lymphocytes de l'humain à des coupes histologiques de tissu utérin ou lymphoïde ou à des homogénats desdits tissus.
PCT/CA2001/001699 2000-11-29 2001-11-29 Methode de controle de l'etat de l'uterus au moyen de cellules tueuses naturelles de l'uterus WO2002044716A1 (fr)

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US7094561B2 (en) * 2000-11-29 2006-08-22 University Of Guelph Method of monitoring the menstrual cycle and/or pregnancy in a female

Non-Patent Citations (6)

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Title
ALI A ASHKAR AND B ANNE CROY: "Functions of uterine natural killer cells are mediated by interferon gamma production during murine pregnancy", IMMUNOLOGY, vol. 13, 2001, pages 235 - 241, XP001057563 *
ASHLEY KING ET AL: "Human Uterine Natural Killer Cells", NATURAL IMMUNITY, vol. 15, 1996 - 1997, pages 41 - 52, XP001057641 *
B A CROY ET AL: "Transplantation into Genetically Alymphoid Mice as an approach to Dissect the Roles of Uterine Natural Killer Cells during Pregnancy-A Review", PLACENTA, vol. 21, no. Sup. A, - March 2000 (2000-03-01), pages s77 - s80, XP001057495 *
B ANNE CROY ET AL: "A study on the density and distributuion of uterine natural killer cells at mid pregnancy in mice", JOURNAL OF REPRODUCTIVE IMMUNOLOGY, vol. 49, 2001, pages 33 - 47, XP000105631 *
B. ANNE CROY ET AL: "Histological studies of gene-ablated mice support important functional roles for natural killer cells in the uterus during pregnancy", JOURNAL OF REPRODUCTIVE IMMUNOLOGY, vol. 35, 1997, pages 111 - 133, XP002192508 *
CHANTARKRU S ET AL: "Contributions from self-renewal and trafficking to the uterine NK cell population of early pregnancy", JOURNAL OF IMMUNOLOGY, vol. 168, no. 1, 1 January 2001 (2001-01-01), pages 22 - 28, XP001057564 *

Cited By (1)

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US7094561B2 (en) * 2000-11-29 2006-08-22 University Of Guelph Method of monitoring the menstrual cycle and/or pregnancy in a female

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