Human Reproduction vol.7 no.6 pp.883-885, 1992
Turner's syndrome patients lack tight junctions between uterine epithelial cells
Monash University Department of Obstetrics and Gynaecology, Monash Medical Centre, 246 Clayton Road, Clayton, Victoria, 3168 and 2Department of Histology and Embryology, University of Sydney, Sydney, N.S.W., 2006, Australia 'To whom correspondence should be addressed
Eleven endometrial biopsies, taken from six Turner's syndrome patients receiving hormone replacement therapy prior to treatment by oocyte donation and embryo transfer, were assessed by freeze fracture followed by electron microscopy for epithelial tight junctions. Nine of the eleven biopsies had no discernible tight junctions; the other two biopsies had reduced and disorganized junctional structures. Two patients subsequently became pregnant following embryo transfer. It is concluded that compromised epithelial integrity does not prevent embryo implantation in the human, an observation that is consistent with a barrier role for the epithelium except at times when appropriately conditioned with oestrogen and progesterone to induce receptivity for implantation. Key words: endometrium/epithelium/oocyte junction/Turner's syndrome
donation/tight
Introduction Tight junctions are important ultrastructural features of epithelial cells. They play a major role in joining adjacent cell walls closely together, thus maintaining the integrity of the epithelium as a barrier against the passage of small molecules and fluids between the cells. Tight junctions close off the extracellular space and so allow epithelia to maintain differential microenvironments between the tissues and the lumen of any organ or the outside of the body (Claude and Goodenough, 1973). It is well documented that pre-implantation embryo development can be significantly influenced by the composition of the uterine luminal environment (Weitlauf, 1976). Examples of this include situations where luminal constituents accelerate embryo development (Garrett et al., 1988), where they result in embryonic diapause (Weitlauf, 1976), and where toxic factors result in the rapid demise of the embryo (Psychoyos et al., 1989). Earlier studies by our group in both rats and humans (Murphy et al., 1982, 1992) have demonstrated that tight junction morphology of uterine epithelial cells alters in a predictable © Oxford University Press
manner in response to the hormonal changes leading up to embryo implantation. This observation suggests that changes in tight junction structure may contribute to successful implantation by regulating the uterine luminal contents. It has also been suggested that the endometrial epithelium plays a major role in determining uterine receptivity for implantation, since removal of the epithelium from a non-progestational uterus will overcome the block to implantation which normally occurs (Cowell, 1969). In the present study we report the structure of uterine epithelial tight junctions in 11 endometrial biopsies taken from six Turner's syndrome patients receiving hormone replacement therapy (HRT) prior to treatment by oocyte donation for in-vitro fertilization and embryo transfer (TVF-ET).
Materials and methods Eleven endometrial biopsies were taken during 11 separate HRT cycles from six women, aged 26—36 years, with Turner's syndrome. This study was approved by the Monash University Human Ethics Committee and all subjects gave informed consent before becoming involved in the study. Of the six patients, one was a full Turner's syndrome (patient D, XO) while the other five were mosaics (patient A, karyotype unavailable; patient B, long arm deletion on X, band 22; patient C, 46Xi(Xg)/45X; patient E, XX/XO; patient F, XO/X-isochrome X). Biopsies 1 and 2 were taken from a patient receiving a fixed HRT regime comprising oestradiol valerate 1 mg/day on days 1-5 and 2 7 - 2 8 , 2 mg/day on days 6 - 9 and 14-26, and 6 mg/day on days 10-13. Progesterone at 25 mg was given on day 15, 50 mg on day 16 and 100 mg/day on days 17 to 26. All other biopsies were taken from patients receiving a lowdosage variable-length oestradiol replacement therapy comprising: oestradiol valerate 2 mg/day from day 1 with progesterone starting at 100 mg/day at any time between days 10 and 18 (Leeton et al., 1991). The day on which progesterone commenced was then recorded as day 1 of the luteal phase or day 15 of a standardized 28 day cycle. Endometrial biopsies were taken by routine curettage and divided into two unequal portions. The larger portion, for ultrastructural freeze fracture work, was immediately fixed in 2.5% glutaraldehyde, 2% formaldehyde in 0.1 M cacodylate buffer (pH 7.4) for 1 h. Following this fixation, the tissue was rinsed three times in cacodylate buffer and then incubated at least overnight in 30% glycerol in buffer. Each piece of this tissue was divided into a number of specimens which were then processed for freeze-fracture electron microscopy by routine techniques as already described in detail (Murphy et al., 1982). 883
Downloaded from https://academic.oup.com/humrep/article-abstract/7/6/883/724749 by University of Western Sydney Library user on 12 January 2019
P.A.W.Rogers1, C.R.Murphy2, J.Leeton, M.J.Hosie2 and L.Beaton
P.A.W.Rogers et al.
Results The results of histological dating of the endometrium, and the day of HRT for each biopsy from the six patients in this study are given in Table I. Nine of the eleven biopsies studied displayed no visible tight junctions that could be found by freeze fracture, which is the most usual method for studying these junctions. This was despite exhaustive searches of a minimum of two and up to five specimens from each biopsy, with a minimum of two replicas being examined for each specimen. This represents more searching than has been necessary to find tight junctions of uterine epithelial cells in all of our previous studies (Murphy et al., 1982, 1992). In only one of the three samples examined from biopsy 10, patient E displayed a tiny amount of very disorganized junctions on one cell. Patient A displayed slightly more junctions but again only in one small area of one sample from biopsy 3. Overall, our searching of replicas found either none or very few junctions and those found were disorganized and very dissimilar from tight junctions seen in human uterine epithelial cells in our previous studies (Murphy et al., 1992). Discussion Tight junctions are generally held to function in preventing or reducing the flow of solutes and small molecules between cells (Claude and Goodenough, 1973; Madara, 1990). Thus, they allow epithelia to develop separate microenvironments in the lumen and surrounding tissue. The environment in the lumen of the uterus in rats and humans is actively contributed to by the epithelial cells; these secretory products in the lumen are thought
Table I. Biopsy data showing day of menstrual cycle according to hormone replacement therapy (HRT) and histopalhological dating Patient
D E F
884
Biopsy number
Day of HRT cycle
Histological dating
1 2 3 4 5 6 7 8 9 10 11
23 24 19 19 19 18 20 20 20 18 22
— 17-18 13 13 14 15-16 19-20 17 18-19
to be important in implantation (Weitlauf, 1976; Murphy et al., 1982). From our current observations, we hypothesize that patients with Turner's syndrome would be unable to maintain differential microenvironments between the lumen and surrounding tissues due to a lack of epithelial tight junctions. Thus, it might be predicted that products secreted into the lumen would be diluted by paracellular epithelial flow. Despite this apparent defect in epithelial integrity, two patients from this study (patients A and E) had successful pregnancies in subsequent embryo transfer cycles. Moreover, there are other reports in the literature of successful pregnancies in Turner's patients following HRT and oocyte donation with FVF —ET (Comet et al., 1990). It is interesting to note that the two Turner's syndrome patients who did become pregnant (A and E), were the two who displayed some rare and disorganized tight junctions, compared to a complete absence in the other four subjects. Nonetheless, the endometrial tissue from these two women was still significantly deficient in tight junctions compared to normal endometrium from the same stage of the menstrual cycle (Murphy et al., 1992). The fact that successful pregnancies occur in patients with Turner's syndrome suggests that deficient or absent endometrial epithelial tight junctions do not compromise early embryo development or implantation. From this it may be concluded that, at least in the human, the specific composition of the uterine luminal environment may not be crucial for successful embryo development. This would fit with observations from IVF, where human pre-implantation embryos have been successfully cultured in a wide range of media and conditions. The observation that an apparently compromised epithelium does not appear to interfere with implantation is also consistent with the hypothesis that the uterine epithelium normally acts as a barrier to the embryo, only becoming receptive for implantation when appropriately conditioned with oestradiol and progesterone. Certainly, it would appear that the absence of uterine epithelial tight junctions does not prevent successful pregnancy. The present findings of abnormal endometrial structure in Turner's patients, with no apparent loss of ability to carry a successful pregnancy, follows an earlier report of consistently subnormal serum levels of pregnancy-associated placenta! protein (PP14) in a Turner's syndrome patient who became pregnant following HRT and frozen embryo transfer (Critchley et al., 1990). Using oocyte donation, the occurrence of pregnancies in previously infertile groups such as Turner's syndrome patients now provides a unique opportunity to identify aspects of normal endometrial structure and function that may in fact not be essential for pregnancy to proceed. Such observations can play an important role in developing and changing our understanding of normal and abnormal endometrial function. Finally, it would be of great interest to know whether other epithelial cell layers in Turner's syndrome patients have abnormal or absent tight junctions, and if they do, what the implications are for their specific functions. Acknowledgement This work was funded by National Health and Medical Research Council grant no. 890092.
Downloaded from https://academic.oup.com/humrep/article-abstract/7/6/883/724749 by University of Western Sydney Library user on 12 January 2019
The smaller portion of each biopsy was fixed in 10% buffered formalin and processed for routine histological dating according to the criteria of Noyes et al. (1950). Each specimen was exhaustively searched for tight junctions. When it first became clear that junctions in these patients were few or non-existent, extra specimens were prepared for each patient and searching for junctions using the electron microscope was performed more thoroughly than has previously been found necessary to locate junctions adequately (Murphy et al., 1982, 1992).
Endometrial tight Junctions in Turner's syndrome
References
Downloaded from https://academic.oup.com/humrep/article-abstract/7/6/883/724749 by University of Western Sydney Library user on 12 January 2019
Claude,P. and Goodenough.D.A. (1973) Fracture faces of zonulae occludentes from "tight" and "leaky" epithelia. J. Cell. Biol., 58, 390-400. Comet.D., Alvarez.S., AntoineJ.M., Tibi.C.H., MandelbaumJ., Plachot.M. and Salat-Baroux,J. (1990) Pregnancies following ovum donation in gonadal dysgenesis. Hum. Reprod., 5, 291—293. Cowell.T.P. (1969) Implantation and development of mouse eggs transferred to the uteri of non-progestational mice. J. Reprod. Fertil., 19, 239-245. Critchley,H.O.D., Chard.T., Lieberman.B.A., Buckley,C.H. and Anderson,D.C. (1990) Serum PPM levels in a patient with Turner's syndrome pregnant after frozen embryo transfer. Hum. Reprod., 5, 250-254. GarrettJ.E., Geisert.R.D., Zavy,M.T. and Morgan.G.L. (1988) Evidence for maternal regulation of early conceptus growth and development in beef cattle. J. Reprod. Fertil., 84, 437-446. LeetonJ., Rogers,P., King.C. and Healy.D. (1991) A comparison of pregnancy rates for 131 donor oocyte transfers using either a sequential or fixed regime of steroid replacement therapy. Hum. Reprod., 6, 299-301. Madara.J.L. (1990) Maintenance of the macromolecular barrier at cell extrusion sites in intestinal epithelium: physiological rearrangement of tight junctions. J. Membr. Biol, 116, 177-184. Murphy.C.R., Swift.J.G., Mukherjee,T.M. and Rogers.A.W. (1982) The structure of tight junctions between uterine luminal epithelial cells at different stages of pregnancy in the rat. Cell Tissue Res., 223, 281-286. Murphy.C.R., Rogers.P.A.W., Leeton.J., Hosie.M. and Beaton.L. (1992) Tight junctions of human uterine epithelial cells change during the menstrual cycle: A morphometric study. Acta Anat., in press. Noyes.R.W., Hertig.A.T. and RockJ. (1950) Dating the endometrial biopsy. Fertil. Sterii, 1, 3 - 2 5 . Psychoyos.A., Roche.D. and Gravanis.A. (1989) Is cholic acid responsible for embryo-toxicity of the post-receptive uterine environment? Hum. Reprod., 4, 832-834. Weitlauf.H.M. (1976) Effects of uterine flushings on RNA synthesis by implanting and "delayed implanting" mouse blastocysts in vitro. Biol. Reprod., 14, 566-571. Received on March 20, 1992; accepted on April 13, 1992
885
Turner's syndrome patients lack tight junctions between uterine epithelial cells
Monash University Department of Obstetrics and Gynaecology, Monash Medical Centre, 246 Clayton Road, Clayton, Victoria, 3168 and 2Department of Histology and Embryology, University of Sydney, Sydney, N.S.W., 2006, Australia 'To whom correspondence should be addressed
Eleven endometrial biopsies, taken from six Turner's syndrome patients receiving hormone replacement therapy prior to treatment by oocyte donation and embryo transfer, were assessed by freeze fracture followed by electron microscopy for epithelial tight junctions. Nine of the eleven biopsies had no discernible tight junctions; the other two biopsies had reduced and disorganized junctional structures. Two patients subsequently became pregnant following embryo transfer. It is concluded that compromised epithelial integrity does not prevent embryo implantation in the human, an observation that is consistent with a barrier role for the epithelium except at times when appropriately conditioned with oestrogen and progesterone to induce receptivity for implantation. Key words: endometrium/epithelium/oocyte junction/Turner's syndrome
donation/tight
Introduction Tight junctions are important ultrastructural features of epithelial cells. They play a major role in joining adjacent cell walls closely together, thus maintaining the integrity of the epithelium as a barrier against the passage of small molecules and fluids between the cells. Tight junctions close off the extracellular space and so allow epithelia to maintain differential microenvironments between the tissues and the lumen of any organ or the outside of the body (Claude and Goodenough, 1973). It is well documented that pre-implantation embryo development can be significantly influenced by the composition of the uterine luminal environment (Weitlauf, 1976). Examples of this include situations where luminal constituents accelerate embryo development (Garrett et al., 1988), where they result in embryonic diapause (Weitlauf, 1976), and where toxic factors result in the rapid demise of the embryo (Psychoyos et al., 1989). Earlier studies by our group in both rats and humans (Murphy et al., 1982, 1992) have demonstrated that tight junction morphology of uterine epithelial cells alters in a predictable © Oxford University Press
manner in response to the hormonal changes leading up to embryo implantation. This observation suggests that changes in tight junction structure may contribute to successful implantation by regulating the uterine luminal contents. It has also been suggested that the endometrial epithelium plays a major role in determining uterine receptivity for implantation, since removal of the epithelium from a non-progestational uterus will overcome the block to implantation which normally occurs (Cowell, 1969). In the present study we report the structure of uterine epithelial tight junctions in 11 endometrial biopsies taken from six Turner's syndrome patients receiving hormone replacement therapy (HRT) prior to treatment by oocyte donation for in-vitro fertilization and embryo transfer (TVF-ET).
Materials and methods Eleven endometrial biopsies were taken during 11 separate HRT cycles from six women, aged 26—36 years, with Turner's syndrome. This study was approved by the Monash University Human Ethics Committee and all subjects gave informed consent before becoming involved in the study. Of the six patients, one was a full Turner's syndrome (patient D, XO) while the other five were mosaics (patient A, karyotype unavailable; patient B, long arm deletion on X, band 22; patient C, 46Xi(Xg)/45X; patient E, XX/XO; patient F, XO/X-isochrome X). Biopsies 1 and 2 were taken from a patient receiving a fixed HRT regime comprising oestradiol valerate 1 mg/day on days 1-5 and 2 7 - 2 8 , 2 mg/day on days 6 - 9 and 14-26, and 6 mg/day on days 10-13. Progesterone at 25 mg was given on day 15, 50 mg on day 16 and 100 mg/day on days 17 to 26. All other biopsies were taken from patients receiving a lowdosage variable-length oestradiol replacement therapy comprising: oestradiol valerate 2 mg/day from day 1 with progesterone starting at 100 mg/day at any time between days 10 and 18 (Leeton et al., 1991). The day on which progesterone commenced was then recorded as day 1 of the luteal phase or day 15 of a standardized 28 day cycle. Endometrial biopsies were taken by routine curettage and divided into two unequal portions. The larger portion, for ultrastructural freeze fracture work, was immediately fixed in 2.5% glutaraldehyde, 2% formaldehyde in 0.1 M cacodylate buffer (pH 7.4) for 1 h. Following this fixation, the tissue was rinsed three times in cacodylate buffer and then incubated at least overnight in 30% glycerol in buffer. Each piece of this tissue was divided into a number of specimens which were then processed for freeze-fracture electron microscopy by routine techniques as already described in detail (Murphy et al., 1982). 883
Downloaded from https://academic.oup.com/humrep/article-abstract/7/6/883/724749 by University of Western Sydney Library user on 12 January 2019
P.A.W.Rogers1, C.R.Murphy2, J.Leeton, M.J.Hosie2 and L.Beaton
P.A.W.Rogers et al.
Results The results of histological dating of the endometrium, and the day of HRT for each biopsy from the six patients in this study are given in Table I. Nine of the eleven biopsies studied displayed no visible tight junctions that could be found by freeze fracture, which is the most usual method for studying these junctions. This was despite exhaustive searches of a minimum of two and up to five specimens from each biopsy, with a minimum of two replicas being examined for each specimen. This represents more searching than has been necessary to find tight junctions of uterine epithelial cells in all of our previous studies (Murphy et al., 1982, 1992). In only one of the three samples examined from biopsy 10, patient E displayed a tiny amount of very disorganized junctions on one cell. Patient A displayed slightly more junctions but again only in one small area of one sample from biopsy 3. Overall, our searching of replicas found either none or very few junctions and those found were disorganized and very dissimilar from tight junctions seen in human uterine epithelial cells in our previous studies (Murphy et al., 1992). Discussion Tight junctions are generally held to function in preventing or reducing the flow of solutes and small molecules between cells (Claude and Goodenough, 1973; Madara, 1990). Thus, they allow epithelia to develop separate microenvironments in the lumen and surrounding tissue. The environment in the lumen of the uterus in rats and humans is actively contributed to by the epithelial cells; these secretory products in the lumen are thought
Table I. Biopsy data showing day of menstrual cycle according to hormone replacement therapy (HRT) and histopalhological dating Patient
D E F
884
Biopsy number
Day of HRT cycle
Histological dating
1 2 3 4 5 6 7 8 9 10 11
23 24 19 19 19 18 20 20 20 18 22
— 17-18 13 13 14 15-16 19-20 17 18-19
to be important in implantation (Weitlauf, 1976; Murphy et al., 1982). From our current observations, we hypothesize that patients with Turner's syndrome would be unable to maintain differential microenvironments between the lumen and surrounding tissues due to a lack of epithelial tight junctions. Thus, it might be predicted that products secreted into the lumen would be diluted by paracellular epithelial flow. Despite this apparent defect in epithelial integrity, two patients from this study (patients A and E) had successful pregnancies in subsequent embryo transfer cycles. Moreover, there are other reports in the literature of successful pregnancies in Turner's patients following HRT and oocyte donation with FVF —ET (Comet et al., 1990). It is interesting to note that the two Turner's syndrome patients who did become pregnant (A and E), were the two who displayed some rare and disorganized tight junctions, compared to a complete absence in the other four subjects. Nonetheless, the endometrial tissue from these two women was still significantly deficient in tight junctions compared to normal endometrium from the same stage of the menstrual cycle (Murphy et al., 1992). The fact that successful pregnancies occur in patients with Turner's syndrome suggests that deficient or absent endometrial epithelial tight junctions do not compromise early embryo development or implantation. From this it may be concluded that, at least in the human, the specific composition of the uterine luminal environment may not be crucial for successful embryo development. This would fit with observations from IVF, where human pre-implantation embryos have been successfully cultured in a wide range of media and conditions. The observation that an apparently compromised epithelium does not appear to interfere with implantation is also consistent with the hypothesis that the uterine epithelium normally acts as a barrier to the embryo, only becoming receptive for implantation when appropriately conditioned with oestradiol and progesterone. Certainly, it would appear that the absence of uterine epithelial tight junctions does not prevent successful pregnancy. The present findings of abnormal endometrial structure in Turner's patients, with no apparent loss of ability to carry a successful pregnancy, follows an earlier report of consistently subnormal serum levels of pregnancy-associated placenta! protein (PP14) in a Turner's syndrome patient who became pregnant following HRT and frozen embryo transfer (Critchley et al., 1990). Using oocyte donation, the occurrence of pregnancies in previously infertile groups such as Turner's syndrome patients now provides a unique opportunity to identify aspects of normal endometrial structure and function that may in fact not be essential for pregnancy to proceed. Such observations can play an important role in developing and changing our understanding of normal and abnormal endometrial function. Finally, it would be of great interest to know whether other epithelial cell layers in Turner's syndrome patients have abnormal or absent tight junctions, and if they do, what the implications are for their specific functions. Acknowledgement This work was funded by National Health and Medical Research Council grant no. 890092.
Downloaded from https://academic.oup.com/humrep/article-abstract/7/6/883/724749 by University of Western Sydney Library user on 12 January 2019
The smaller portion of each biopsy was fixed in 10% buffered formalin and processed for routine histological dating according to the criteria of Noyes et al. (1950). Each specimen was exhaustively searched for tight junctions. When it first became clear that junctions in these patients were few or non-existent, extra specimens were prepared for each patient and searching for junctions using the electron microscope was performed more thoroughly than has previously been found necessary to locate junctions adequately (Murphy et al., 1982, 1992).
Endometrial tight Junctions in Turner's syndrome
References
Downloaded from https://academic.oup.com/humrep/article-abstract/7/6/883/724749 by University of Western Sydney Library user on 12 January 2019
Claude,P. and Goodenough.D.A. (1973) Fracture faces of zonulae occludentes from "tight" and "leaky" epithelia. J. Cell. Biol., 58, 390-400. Comet.D., Alvarez.S., AntoineJ.M., Tibi.C.H., MandelbaumJ., Plachot.M. and Salat-Baroux,J. (1990) Pregnancies following ovum donation in gonadal dysgenesis. Hum. Reprod., 5, 291—293. Cowell.T.P. (1969) Implantation and development of mouse eggs transferred to the uteri of non-progestational mice. J. Reprod. Fertil., 19, 239-245. Critchley,H.O.D., Chard.T., Lieberman.B.A., Buckley,C.H. and Anderson,D.C. (1990) Serum PPM levels in a patient with Turner's syndrome pregnant after frozen embryo transfer. Hum. Reprod., 5, 250-254. GarrettJ.E., Geisert.R.D., Zavy,M.T. and Morgan.G.L. (1988) Evidence for maternal regulation of early conceptus growth and development in beef cattle. J. Reprod. Fertil., 84, 437-446. LeetonJ., Rogers,P., King.C. and Healy.D. (1991) A comparison of pregnancy rates for 131 donor oocyte transfers using either a sequential or fixed regime of steroid replacement therapy. Hum. Reprod., 6, 299-301. Madara.J.L. (1990) Maintenance of the macromolecular barrier at cell extrusion sites in intestinal epithelium: physiological rearrangement of tight junctions. J. Membr. Biol, 116, 177-184. Murphy.C.R., Swift.J.G., Mukherjee,T.M. and Rogers.A.W. (1982) The structure of tight junctions between uterine luminal epithelial cells at different stages of pregnancy in the rat. Cell Tissue Res., 223, 281-286. Murphy.C.R., Rogers.P.A.W., Leeton.J., Hosie.M. and Beaton.L. (1992) Tight junctions of human uterine epithelial cells change during the menstrual cycle: A morphometric study. Acta Anat., in press. Noyes.R.W., Hertig.A.T. and RockJ. (1950) Dating the endometrial biopsy. Fertil. Sterii, 1, 3 - 2 5 . Psychoyos.A., Roche.D. and Gravanis.A. (1989) Is cholic acid responsible for embryo-toxicity of the post-receptive uterine environment? Hum. Reprod., 4, 832-834. Weitlauf.H.M. (1976) Effects of uterine flushings on RNA synthesis by implanting and "delayed implanting" mouse blastocysts in vitro. Biol. Reprod., 14, 566-571. Received on March 20, 1992; accepted on April 13, 1992
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