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The effect of the oviduct, uterine, and in vitro environments on zona thinning in the mouse embryo
Reproductive animal research
FERTILITY AND STERILITY@
Vol. 68. No.1. July 1997
Copyright" 1997 American Society for Reproductive Medicine
Printed on acid-free paper in U. S. A.
Published by Elsevier Science Inc.
The effect of the oviduct, uterine, and in vitro environments on zona thinning in the mouse embryo Edmond Confine, M.D. * Richard Rawlins, Ph.D. Zvi Binor, M.D. Ewa Radwanska, M.D., Ph.D. Division of Reproductive Endocrinology and Infertility, Rush Presbyterian St. Luke's Medical Center, Chicago, Illinois
Objective: To evaluate the impact of the oviduct, uterine, and in vitro environments on zona pellucida thinning in the mouse embryo. Design: Female mice were stimulated with pregnant mare serum gonadotropin and mated after hCG injection. Unilateral oviduct ligation was performed on day 2 of gestation using the dorsal approach. The mice were divided into equal groups and killed on days 2, 3, 4, 5, and 10 of gestation. In vitro incubated embryos served as controls. Average daily zona thickness measurements were subjected to analysis of variance and paired Student's t-test. Setting: The laboratory of the assisted reproductive program of Rush University Medical Center. Main Outcome Measure(s): Progressive daily decrease in average zona thickness. ResuIt(s): Zona measurements of embryos flushed out of uterine horns, ligated oviducts, and in vitro incubation demonstrated statistically significant decreases in zona thickness, from 9.6 ± 1.6 p,m (day 3) to 6.0 ± 0.8 p,m (day 5), from 11.6 ± 2.2 p,m (day 2) to 6.0 ± 1.6 p,m (day 5), and from 11.1 ± 2.0 p,m (day 2) to 6.0 ± 1.6 p,m (day 5), respectively. There were no differences in average zona thickness for embryos in the same cell stage and same protocol day in all three locations. Conclusion(s): Zona thinning seems to be induced primarily by the dividing embryo before implantation. A substantial tubal and uterine contribution to zona thinning was not detected in this mouse embryo model. (Fertil Sterilv 1997;68:164-7. © 1997 by American Society for Reproductive Medicine.) Key Words: Embryo, zona pellucida, oviduct, uterus
The zona pellucida (ZP) maintains the integrity of the early embryo. In the absence of the zona, the embryos disperse and pregnancy does not occur (1, 2). Zona-free embryos adhere to the oviductal walls, and embryo cleavage is inhibited in zona-free blastomeres (2). Implantation occurs only after the transfer of zona-free compacted embryos at the morula and blastocyst stages, after formation of intercellular bridges (3, 4). Received February 5, 1996; revised and accepted March 13, 1997. * Reprint requests and present address: Edmond Confino, M.D., Northwestern University Hospital, 333 East Superior Street, Chicago, Illinois 60611 (FAX: 312-908-6643).
164
Thinning of the zona occurs in most mammals before hatching. The zona progressively decreases in thickness until an area in the zona disrupts and the embryo hatches through the gap, typically at the blastocyst stage. It is believed that zona thinning is affected by the dividing embryo, which exerts a mechanical stretch on the zona (5). Support of the mechanical theory of zona thinning comes from the observation that embryos with perforated zonae do not achieve zona thinning (6). Zona thinning also may be affected by the tubal and uterine environments (7, 8). Mouse embryo implantation fails after ovariectomy and lactation. Delayed hatching is observed and the zonae do not thin. Once implantation occurs, lysis of the zonae is observed (9). Therefore, 0015-0282/97/$17.00 PH SOOI5-0282(97)00080-0
zona lysis in vivo may be dependent on both uterine and embryonal factors. The assumption is that the embryo signals to the uterus, which secretes zona lytic materials. The presence of putative zona lysins of embryonal origin is supported by the observation that the incubation of embryos in groups enhances hatching in vitro. Embryos hatch poorly in vitro if they are cultured separately, suggesting that mouse embryos secrete zona lysing materials (10). This putative zona lytic substance may have chymotrypsin-like activity (11- 13). Defective zona thinning and subsequently impaired embryo hatching may contribute to failed implantation in some apparently normal human embryos. Zona manipulations developed to correct defective zona thinning include chemical digestion and laser and mechanical disruption ofthe zona (12, 14, 15). Timed and gentle zona micromanipulation obviously is important to avoid damage to the embryo, premature hatching, and dispersion of the blastomeres. The concept of assisted hatching was developed to enhance implantation, typically in older patients (14) considered to have a poor prognosis for IVF (15). The purpose of this study was to test whether mouse zona thinning is a centrifugal event induced by the dividing embryo or a centripetal event in duced by the tubal and uterine environments, or both. MATERIALS AND METHODS
Experiments were performed on 36 virgin female mice, 7 weeks old, of strain CBCFI. The female mice were mated with four male mice of known, good reproductive performance. The female mice were injected intraperitoneally with 10 IV of pregnant mare serum gonadotropin. Two days later, they were injected with 100 IV of hCG and mated with a male mouse . A vaginal mucus plug was detected during the following 36 hours (day 1 of gestation). Two mice that did not conceive were excluded from the study, and two mice were used to practice oviduct flushing and oviduct ligation. Twenty mice underwent unilateral oviduct ligation via a dorsal approach on day 2 of gestation. Ether inhalation anesthesia was used during oviduct ligation. After dorsal entry into the abdominal cavity , the ovary , uterine horn, and oviduct were identified, and a 6/0 ligature was placed between the uterine horn and the mouse oviduct. The abdominal wall was closed in two layers. The mice were observed until complete recovery and were transferred to their cages. Four mice were killed by cervical dislocation on days 2, 3, 4, and 5 of gestation. The uterVol. 68, No. I, July 1997
ine horns and oviducts were dissected and flushed with protein-supplemented Ham's F-10 medium (GIBCO, Grand Island, N"Y) under a stereomicroscope. The embryos were extracted with a micropipette, transferred to a culture dish, and incubated at 37°C in a gas-controlled incubator with 5% CO2 and room air. Four equatorial zona measurements of wet-mounted embryos of the same cell stage were obtained and averaged using a calibrated invertedphase microscope. Embryos with cell stage that did not match the protocol day were not included in the study. Four mice that underwent oviduct ligation were killed on day 10 of gestation. The oviduct and the uterine hom were inspected for gestational sacs under a stereomicroscope, and pregnancy was confirmed pathologically. Ten mice were killed on day 2 of gestation, and 78 embryos were flushed out of the oviducts and cultured for 4 days. Daily zona measurements were obtained as described earlier. Abnormal, compacted, and degenerating embryos were excluded from the study (Fig. 1). The data were subjected to analysis of variance and paired Student's t-tests.
RESULTS No embryos were retrieved from the ligated uteri. The ligated oviducts yielded 41, 32, 31, and 28 embryos of the same cell stage on gestation days 2 through 5, respectively. The unligated oviducts yielded 35 embryos at the two- to four-cell stage on day 2 of gestation. Vnligated uteri yielded 36, 35, and 34 embryos of the same cell stage on gestation days 3 through 5, respectively. Seventy-eight embryos with two to four cells obtained from the oviducts of 10 mice on day 2 were incubated in vitro, and the zonae were measured daily.
Figure 1 A normal mous e blastocyst (bottom ) and a degenerating mou se embryo (top ) flush ed out of the uterus on day 5 of gestation. The zona of th e degener ating embryo is thicker th an th e zona of the normal blastocyst . Confino et al.
Zona thinning
165
Table 1 Average Zona Thickness Measu rements of Embryos Flu shed From Ligated and Unligat ed Oviducts and Uteri, and Embryos Incubated In Vit ro*
Ligated oviduct Unligate d oviduct Unligated uterus In vit ro incubation
Day 2 (2 to 4 cells)
Day 3 (morula)
Day 4 (early blastocyst)
Day 5 (late blastocyst)
11.6 ± 2.2 12.1 ± 2.4
8.4 ± 2.0
7.6 ± 1.6
5.6 ± 1.6
9.6 ± 1.6 8.4 ± 1.2
6.8 ± 1.0 6.4 ± 0.8
6.0 ± 0.8 6.0 ± 1.6
11.1 ± 2.0
* Values ar e means ± SD, in ~m . Analysis of variance of zona thickness done separately for each day on ligated and unligated oviduct and unligated uterus did not demonstrate statistica lly significant differences . Pa ired Student's z-test (day 2 ver sus day 5) demonstrated P < 0.05 for ligat ed oviduct , in vitro incubation, unligated oviduct, and unligated uterus (day 3 versus day 5 for th e latter one).
Table 1 depicts changes in zona thickness in samecell-stage embryos obtained from oviducts, uteri, and in vitro incubation. Average zona thickness of embryos flushed from the corresponding ligated oviducts decreased in 4 days from 11.6 ± 2.2 to 5.6 ± 1.6 /-lm (P < 0.05). The average zona thickness of embryos obtained from the unligated uterus decreased from 9.6 ± 1.6 to 6.0 ± 0.8 /-lm (P < 0.05). Embryos incubated in vitro from day 2 through day 5 of gestation demonstrated a decrease in average zona thickness from 11.6 ± 2.0 to 6.0 ± 1.6 /.Lm (P < 0.05). Analysis of variance of the average zona thickness of same-cell-stage embryos did not reveal any statistically significant differences in all three locations or in vitro . Two mice developed what seemed to be an oviductal gestation adjacent to the tubal ligature (Fig. 2), detected on day 10 of gestation , and normal gestations in the unligated contralateral uterine horns. DISCUSSION
The research of zona changes improves our understanding of events occurring before implantation. Und erstandably, zona research in the human model
Figure 2 An oviductal gestation (day 10 of gestation). The arrow depicts the oviductal st itch groove. 166
Confino et al, Zona th inning
would be ethically difficult to perform. Zona studies in a rodent model provide valuable information, but they cannot be extrapolated to the human species . This study was conceived because of increasing interest in the role of the ZP in IVF implantation failure s (14). Assisted hatching procedures using partial zona dissection or chemical zona digestion are still in their inception. Assisted hatching assumes that the embryo is normal bu t incapable of thinning the zona, especially in older patients who fail IVF (15). Zona hardening is associated with advanced patient age, in vitro incubation in a culture medium, and impaired hatching (16). In t heory, accelerated zona thinning may result in premature hatching and the development of tubal pregnancy. The results of this study suggest that implantation can occur occasionally in t he mouse oviduct when oviductal continuity is interrupted (Fig. 2). The se implantations occurred adjacent to the oviduct ligation stitch. It is possible that embryos implanted in the oviduct, or they implanted on a small extension of uterine mucosa beyond the ligature. This observation was surprising and unexpected because of practically nonexistent oviductal gestations in the rodent. Variations in the accuracy of microplacement of ligatures limit our ability to speculate on the importance of this findin g. Zona thinning presumably occurs because of enzymatic digestion of the trophoblast, mech anical stretching of the enlarging blastocyst, and potential effects of the uterus and oviduct. This study was designed to answer whether zona thinning is a cent rifugal, embryo-induced event or whether the tubal and uterine environments also contribute to zona thinning. The contralateral normal uterine horn in each mouse served as a control for the ligated uterine horn . Zonae were measured only in embryos of the same cell stage and same protocol day to control for the expected variability in conception time. Four equatorial zona measurements allowed close observation of the entire circumference of the embryo . Embryos were wet mounted to eliminate compression artifacts. Fertility and St erilityv
This study demonstrated that the zonae thinned progressively in embryos retained within the oviduct, the uterine horn, and in vitro (Table 1). Statistical analysis did not reveal any significant differences in zona thickness when the oviduct, uterus, and in vitro culture environments were compared in same-cell-stage embryos. The number of mice used could not detect very small differences in zona thinning. However, very small differences in zona thinning in these locations were oflittle biologic interest. Therefore, expanding the study and including more animals to achieve more statistical power seemed unjustified. Interpretation of the data assumed that if the uterine environment had a significant contribution to zona thinning, the embryos incubated in vitro would have had significantly thicker zonae compared with same-cell-stage embryos retrieved from the uterine horns. The observation that zona thickness was not different in these locations suggested that the contribution of the uterine and oviductal environments to zona thinning probably was of small biologic magnitude before implantation. Therefore, we concluded that the dividing embryo primarily contributed to zona thinning. The hypothesis that the uterine and oviductal environments contribute significantly to zona thinning before implantation was not supported by these results. If zona thinning is primarily a centrifugal event induced by the dividing embryo, then the normality of the dividing embryo is the most important determinant of zona thinning and subsequent embryo hatching. It was reported that the presence of excessive amounts of degenerate material in the embryo, indicative of poor embryo quality, impaired the hatching process (17). This study confirmed that abnormal embryos arrested at the morula stage retained thicker zonae compared with embryos that kept dividing to the blastocyst stage (Fig. 1). This observation supports the contention that the normality of the dividing embryo is essential to normal zona thinning.
Vol. 68, No.1, July 1997
REFERENCES 1. Edwards RG. Cleavage of one- and two-celled rabbit eggs in vitro after removal of the zona pellucida. J Reprod Fertil 1964;7:413-5. 2. Modlinski JA. The role of the zona pellucida in the development of mouse eggs in vitro. J Embryol Exp Morphol 1979;23:539-47. 3. Bronson RA, McLaren A. Transfer to the mouse oviduct of eggs with and without the zona pellucida. J Reprod Fertil 1970;22:129-37. 4. Trounson AO, Moore NW. The survival and development of sheep eggs following complete or partial removal of the zona pellucida. J Reprod Fertil1974;41:97-105. 5. Cole RJ. Cinemicrographic observation on the trophoblast and zona pellucida of the mouse blastocyst. J Embryol Exp MorphoI1967;17:481-90. 6. Nieder GL, Caprio TL. Early development in the Siberian hamster (Phodopus sungorous). Mol Reprod Dev 1990;27: 224-9. 7. McLaren A. The fate of the zona pellucida in mice. J Embryol Exp MorphoI1970;23:1-19. 8. Mintz B. Control of embryo implantation and survival. Adv Biosci 1971;6:317-42. 9. McLaren A. A study of blastocysts during delay and subsequent implantation in lactating mice. J Endocrinol 1968; 42:453-63. 10. Wright RW, Watson JG, Chaykin S. Factors influencing the in vitro hatching of mouse blastocysts. Anim Reprod Sci 1978; 1:181-8. 11. Yasumasu S, Katow S, Umino Y, Iuchi I, Yamagami K. A unique proteolytic action of HCE, a constituent protease of a fish hatching enzyme. Tight binding to its natural substrate, egg envelope. Biochem Biophys Res Commun 1989;161:58-63. 12. Lepage T, Gache C. Purification and characterization of the sea urchin embryo hatching enzyme. J BioI Chem 1989;262: 4787-93. 13. Post LL, Schuel R, Schuel H. Evidence that hatching enzyme of the sea urchin Stronylocentrotus pupuratus is a chymotrypsin-like protease. Biochem Cell BioI 1988; 66:1200-9. 14. Katayama KP. Assisted hatching: the current status and future projections. Assist Reprod Rev 1994;4:33-8. 15. Schoolcraft WB, Schlenker T, Gee M, Jones GS, Jones HW. Assisted hatching in the treatment of poor prognosis in vitro fertilization candidates. Fertil SteriI1994;62:551-4. 16. DeFelici M, Siracusa G. "Spontaneous" hardening of the zona pellucida of mouse oocytes during in vitro culture. Gamete Res 1982;6:107-13. 17. Cohen J, Alikani M, Liu HC, Rosenwaks Z. Rescue of human embryos by micromanipulation [review]. Baillieres Clin Obstet Gynaecol 1994;8:95-116.
Confino et al.
Zona thinning
167
FERTILITY AND STERILITY@
Vol. 68. No.1. July 1997
Copyright" 1997 American Society for Reproductive Medicine
Printed on acid-free paper in U. S. A.
Published by Elsevier Science Inc.
The effect of the oviduct, uterine, and in vitro environments on zona thinning in the mouse embryo Edmond Confine, M.D. * Richard Rawlins, Ph.D. Zvi Binor, M.D. Ewa Radwanska, M.D., Ph.D. Division of Reproductive Endocrinology and Infertility, Rush Presbyterian St. Luke's Medical Center, Chicago, Illinois
Objective: To evaluate the impact of the oviduct, uterine, and in vitro environments on zona pellucida thinning in the mouse embryo. Design: Female mice were stimulated with pregnant mare serum gonadotropin and mated after hCG injection. Unilateral oviduct ligation was performed on day 2 of gestation using the dorsal approach. The mice were divided into equal groups and killed on days 2, 3, 4, 5, and 10 of gestation. In vitro incubated embryos served as controls. Average daily zona thickness measurements were subjected to analysis of variance and paired Student's t-test. Setting: The laboratory of the assisted reproductive program of Rush University Medical Center. Main Outcome Measure(s): Progressive daily decrease in average zona thickness. ResuIt(s): Zona measurements of embryos flushed out of uterine horns, ligated oviducts, and in vitro incubation demonstrated statistically significant decreases in zona thickness, from 9.6 ± 1.6 p,m (day 3) to 6.0 ± 0.8 p,m (day 5), from 11.6 ± 2.2 p,m (day 2) to 6.0 ± 1.6 p,m (day 5), and from 11.1 ± 2.0 p,m (day 2) to 6.0 ± 1.6 p,m (day 5), respectively. There were no differences in average zona thickness for embryos in the same cell stage and same protocol day in all three locations. Conclusion(s): Zona thinning seems to be induced primarily by the dividing embryo before implantation. A substantial tubal and uterine contribution to zona thinning was not detected in this mouse embryo model. (Fertil Sterilv 1997;68:164-7. © 1997 by American Society for Reproductive Medicine.) Key Words: Embryo, zona pellucida, oviduct, uterus
The zona pellucida (ZP) maintains the integrity of the early embryo. In the absence of the zona, the embryos disperse and pregnancy does not occur (1, 2). Zona-free embryos adhere to the oviductal walls, and embryo cleavage is inhibited in zona-free blastomeres (2). Implantation occurs only after the transfer of zona-free compacted embryos at the morula and blastocyst stages, after formation of intercellular bridges (3, 4). Received February 5, 1996; revised and accepted March 13, 1997. * Reprint requests and present address: Edmond Confino, M.D., Northwestern University Hospital, 333 East Superior Street, Chicago, Illinois 60611 (FAX: 312-908-6643).
164
Thinning of the zona occurs in most mammals before hatching. The zona progressively decreases in thickness until an area in the zona disrupts and the embryo hatches through the gap, typically at the blastocyst stage. It is believed that zona thinning is affected by the dividing embryo, which exerts a mechanical stretch on the zona (5). Support of the mechanical theory of zona thinning comes from the observation that embryos with perforated zonae do not achieve zona thinning (6). Zona thinning also may be affected by the tubal and uterine environments (7, 8). Mouse embryo implantation fails after ovariectomy and lactation. Delayed hatching is observed and the zonae do not thin. Once implantation occurs, lysis of the zonae is observed (9). Therefore, 0015-0282/97/$17.00 PH SOOI5-0282(97)00080-0
zona lysis in vivo may be dependent on both uterine and embryonal factors. The assumption is that the embryo signals to the uterus, which secretes zona lytic materials. The presence of putative zona lysins of embryonal origin is supported by the observation that the incubation of embryos in groups enhances hatching in vitro. Embryos hatch poorly in vitro if they are cultured separately, suggesting that mouse embryos secrete zona lysing materials (10). This putative zona lytic substance may have chymotrypsin-like activity (11- 13). Defective zona thinning and subsequently impaired embryo hatching may contribute to failed implantation in some apparently normal human embryos. Zona manipulations developed to correct defective zona thinning include chemical digestion and laser and mechanical disruption ofthe zona (12, 14, 15). Timed and gentle zona micromanipulation obviously is important to avoid damage to the embryo, premature hatching, and dispersion of the blastomeres. The concept of assisted hatching was developed to enhance implantation, typically in older patients (14) considered to have a poor prognosis for IVF (15). The purpose of this study was to test whether mouse zona thinning is a centrifugal event induced by the dividing embryo or a centripetal event in duced by the tubal and uterine environments, or both. MATERIALS AND METHODS
Experiments were performed on 36 virgin female mice, 7 weeks old, of strain CBCFI. The female mice were mated with four male mice of known, good reproductive performance. The female mice were injected intraperitoneally with 10 IV of pregnant mare serum gonadotropin. Two days later, they were injected with 100 IV of hCG and mated with a male mouse . A vaginal mucus plug was detected during the following 36 hours (day 1 of gestation). Two mice that did not conceive were excluded from the study, and two mice were used to practice oviduct flushing and oviduct ligation. Twenty mice underwent unilateral oviduct ligation via a dorsal approach on day 2 of gestation. Ether inhalation anesthesia was used during oviduct ligation. After dorsal entry into the abdominal cavity , the ovary , uterine horn, and oviduct were identified, and a 6/0 ligature was placed between the uterine horn and the mouse oviduct. The abdominal wall was closed in two layers. The mice were observed until complete recovery and were transferred to their cages. Four mice were killed by cervical dislocation on days 2, 3, 4, and 5 of gestation. The uterVol. 68, No. I, July 1997
ine horns and oviducts were dissected and flushed with protein-supplemented Ham's F-10 medium (GIBCO, Grand Island, N"Y) under a stereomicroscope. The embryos were extracted with a micropipette, transferred to a culture dish, and incubated at 37°C in a gas-controlled incubator with 5% CO2 and room air. Four equatorial zona measurements of wet-mounted embryos of the same cell stage were obtained and averaged using a calibrated invertedphase microscope. Embryos with cell stage that did not match the protocol day were not included in the study. Four mice that underwent oviduct ligation were killed on day 10 of gestation. The oviduct and the uterine hom were inspected for gestational sacs under a stereomicroscope, and pregnancy was confirmed pathologically. Ten mice were killed on day 2 of gestation, and 78 embryos were flushed out of the oviducts and cultured for 4 days. Daily zona measurements were obtained as described earlier. Abnormal, compacted, and degenerating embryos were excluded from the study (Fig. 1). The data were subjected to analysis of variance and paired Student's t-tests.
RESULTS No embryos were retrieved from the ligated uteri. The ligated oviducts yielded 41, 32, 31, and 28 embryos of the same cell stage on gestation days 2 through 5, respectively. The unligated oviducts yielded 35 embryos at the two- to four-cell stage on day 2 of gestation. Vnligated uteri yielded 36, 35, and 34 embryos of the same cell stage on gestation days 3 through 5, respectively. Seventy-eight embryos with two to four cells obtained from the oviducts of 10 mice on day 2 were incubated in vitro, and the zonae were measured daily.
Figure 1 A normal mous e blastocyst (bottom ) and a degenerating mou se embryo (top ) flush ed out of the uterus on day 5 of gestation. The zona of th e degener ating embryo is thicker th an th e zona of the normal blastocyst . Confino et al.
Zona thinning
165
Table 1 Average Zona Thickness Measu rements of Embryos Flu shed From Ligated and Unligat ed Oviducts and Uteri, and Embryos Incubated In Vit ro*
Ligated oviduct Unligate d oviduct Unligated uterus In vit ro incubation
Day 2 (2 to 4 cells)
Day 3 (morula)
Day 4 (early blastocyst)
Day 5 (late blastocyst)
11.6 ± 2.2 12.1 ± 2.4
8.4 ± 2.0
7.6 ± 1.6
5.6 ± 1.6
9.6 ± 1.6 8.4 ± 1.2
6.8 ± 1.0 6.4 ± 0.8
6.0 ± 0.8 6.0 ± 1.6
11.1 ± 2.0
* Values ar e means ± SD, in ~m . Analysis of variance of zona thickness done separately for each day on ligated and unligated oviduct and unligated uterus did not demonstrate statistica lly significant differences . Pa ired Student's z-test (day 2 ver sus day 5) demonstrated P < 0.05 for ligat ed oviduct , in vitro incubation, unligated oviduct, and unligated uterus (day 3 versus day 5 for th e latter one).
Table 1 depicts changes in zona thickness in samecell-stage embryos obtained from oviducts, uteri, and in vitro incubation. Average zona thickness of embryos flushed from the corresponding ligated oviducts decreased in 4 days from 11.6 ± 2.2 to 5.6 ± 1.6 /-lm (P < 0.05). The average zona thickness of embryos obtained from the unligated uterus decreased from 9.6 ± 1.6 to 6.0 ± 0.8 /-lm (P < 0.05). Embryos incubated in vitro from day 2 through day 5 of gestation demonstrated a decrease in average zona thickness from 11.6 ± 2.0 to 6.0 ± 1.6 /.Lm (P < 0.05). Analysis of variance of the average zona thickness of same-cell-stage embryos did not reveal any statistically significant differences in all three locations or in vitro . Two mice developed what seemed to be an oviductal gestation adjacent to the tubal ligature (Fig. 2), detected on day 10 of gestation , and normal gestations in the unligated contralateral uterine horns. DISCUSSION
The research of zona changes improves our understanding of events occurring before implantation. Und erstandably, zona research in the human model
Figure 2 An oviductal gestation (day 10 of gestation). The arrow depicts the oviductal st itch groove. 166
Confino et al, Zona th inning
would be ethically difficult to perform. Zona studies in a rodent model provide valuable information, but they cannot be extrapolated to the human species . This study was conceived because of increasing interest in the role of the ZP in IVF implantation failure s (14). Assisted hatching procedures using partial zona dissection or chemical zona digestion are still in their inception. Assisted hatching assumes that the embryo is normal bu t incapable of thinning the zona, especially in older patients who fail IVF (15). Zona hardening is associated with advanced patient age, in vitro incubation in a culture medium, and impaired hatching (16). In t heory, accelerated zona thinning may result in premature hatching and the development of tubal pregnancy. The results of this study suggest that implantation can occur occasionally in t he mouse oviduct when oviductal continuity is interrupted (Fig. 2). The se implantations occurred adjacent to the oviduct ligation stitch. It is possible that embryos implanted in the oviduct, or they implanted on a small extension of uterine mucosa beyond the ligature. This observation was surprising and unexpected because of practically nonexistent oviductal gestations in the rodent. Variations in the accuracy of microplacement of ligatures limit our ability to speculate on the importance of this findin g. Zona thinning presumably occurs because of enzymatic digestion of the trophoblast, mech anical stretching of the enlarging blastocyst, and potential effects of the uterus and oviduct. This study was designed to answer whether zona thinning is a cent rifugal, embryo-induced event or whether the tubal and uterine environments also contribute to zona thinning. The contralateral normal uterine horn in each mouse served as a control for the ligated uterine horn . Zonae were measured only in embryos of the same cell stage and same protocol day to control for the expected variability in conception time. Four equatorial zona measurements allowed close observation of the entire circumference of the embryo . Embryos were wet mounted to eliminate compression artifacts. Fertility and St erilityv
This study demonstrated that the zonae thinned progressively in embryos retained within the oviduct, the uterine horn, and in vitro (Table 1). Statistical analysis did not reveal any significant differences in zona thickness when the oviduct, uterus, and in vitro culture environments were compared in same-cell-stage embryos. The number of mice used could not detect very small differences in zona thinning. However, very small differences in zona thinning in these locations were oflittle biologic interest. Therefore, expanding the study and including more animals to achieve more statistical power seemed unjustified. Interpretation of the data assumed that if the uterine environment had a significant contribution to zona thinning, the embryos incubated in vitro would have had significantly thicker zonae compared with same-cell-stage embryos retrieved from the uterine horns. The observation that zona thickness was not different in these locations suggested that the contribution of the uterine and oviductal environments to zona thinning probably was of small biologic magnitude before implantation. Therefore, we concluded that the dividing embryo primarily contributed to zona thinning. The hypothesis that the uterine and oviductal environments contribute significantly to zona thinning before implantation was not supported by these results. If zona thinning is primarily a centrifugal event induced by the dividing embryo, then the normality of the dividing embryo is the most important determinant of zona thinning and subsequent embryo hatching. It was reported that the presence of excessive amounts of degenerate material in the embryo, indicative of poor embryo quality, impaired the hatching process (17). This study confirmed that abnormal embryos arrested at the morula stage retained thicker zonae compared with embryos that kept dividing to the blastocyst stage (Fig. 1). This observation supports the contention that the normality of the dividing embryo is essential to normal zona thinning.
Vol. 68, No.1, July 1997
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