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Clinical parameters influencing human zona pellucida thickness*
FERTILITY AND STERILITY®
Vol. 66, No.3, September 1996
Printed on acid-free paper in U. S. A
Copyright c 1996 American Society for Reproductive Medicine
Clinical parameters influencing human zona pellucida thickness*
Evelyne Bertrand, M.Sc.t Marc Van den Bergh, M.T. Yvon Englert, M.D., Ph.D. Department of Obstetric and Gynecology, Fertility Unit, Free University of Brussels, Erasme Hospital, Brussels, Belgium
Objectives: To assess which clinical parameters influence human oocyte zona pellucida (ZP) thickness. Patients: Sixty-five couples undergoing 75 IVF-ET cycles. Main Outcome Measure: Zona pellucida thickness of 827 oocytes measured 16 to 20 hours after in vitro insemination under inverted light microscope. Results: Zona pellucida thickness was 18.9 ± 3.8 p,m (mean ± SD) for unfertilized, 16.4 ± 3.1 p,m for fertilized, and 15.1 ± 2.4 p,m for polyspermic oocytes (significantly different). Among our patients, a few underwent two (or even three) IVF-ET cycles, and the mean ZP thickness was, in most cases, not significantly different from one cycle to the other(s). Regression analyses were calculated between ZP thickness and available clinical parameters, i.e., the age of the women, the duration of stimulation, the cumulus maturity, the number of retrieved oocytes, the number ofhMG doses, the maximum E 2 level, and the follicular volume. A significant linear decreasing relationship exists between the mean ZP thickness of each patient and the maximum E2 level and an increasing one with the hMG dose. Relationships with the other parameters appeared to be nonsignificant. Conclusion: The ZP thickness is basically an individual feature that influences the fertilization rate. Nevertheless, it may be influenced slightly by the hormonal treatment during stimulation. Fertil Steril® 1996; 66:408-11 Key Words: Fertilization rate, human oocytes, IVF-ET, zona pellucida thickness, hormonal stimulation
Gamete-specific glycoproteins compose the relatively thick acellular glycoprotein coat, the zona pellucida (ZP), and participate in various essential functions of the fertilization process (1, 2). Its thickness varies between mammalian species and also between individuals of a single species. The ZP thickness seems to be correlated directly with protein content (3,4). It generally is considered that fertilization rate in IVF-ET is closely dependent on sperm characteristics. Nevertheless, a previous study on human oocytes fertilized in vitro has shown that ZP thickness has an influence on the fertilization rate because fertilized oocytes have, on average, a significantly thinner ZP than unfertilized oocytes (3). Received August 3,1995; revised and accepted April 11, 1996. * Supported by the Fonds National de Ie Recherche Scientifique, Brussels, Belgium. t Reprint requests: Evelyne Bertrand, M.Sc., Department of Obstetric and Gynecology, Fertility Unit, Erasme Hospital, 808 Route de Lennik, 1070 Brussels, Belgium (FAX.: 32-25556672). 408
Bertrand et al. Zona pellucida thickness
The failure of IVF-ET may be linked to a general oocyte dysfunction, the ZP thickness being only a marker of it. This dysfunction could be intrinsic to the oocyte, to the patient, or linked to clinical characteristics such as hormonal stimulation. The aim of the present study was to assess, in an IVF-ET program, which available clinical parameters could intervene in the ZP thickness. MATERIALS AND METHODS Patient Data
Zona pellucida thickness was measured on oocytes collected during 75 IVF-ET cycles performed in 65 couples. Among those patients, 44 couples suffering only from female infertilities were selected to assess the difference between unfertilized, fertilized (two pronuclei), and polyspermic oocytes (more than two pronuclei). The women ranged in age from 23 to 42 years (34.2 Fertility and Sterility®
± 4.0 years; mean ± SD). The duration of infertility was 6.5 ± 4.1 years. Within the 65 couples, 8 underwent two cycles and 2 underwent three cycles. Treatment Protocol
Mter classical follicular stimulation using hMG (Humegon; Organon, Oss, The Netherlands and Pergonal; Serono, Milan, Italy) under LH-releasing hormone analogue (buserelin acetate, Suprefact; Hoechst Inc., Frankfurt, Germany), oocytes were picked up under transvaginal ultrasonic guidance, 36 hours after ovulation induction by 104 IU hCG (Profasi; Serono and Pregnyl; Organon). Follicles were aspirated and the volume of follicular fluid obtained from each aspirated follicle was measured as described by Arnot et al. (5). The mean follicular volume was 3.7 ± 2.0 mL. The mean maximum E2 level in the peripheral plasma was evaluated by RIAs and reached 2.6 ± 1.1 ng/mL (conversion factor to SI unit, 3.671). The mean number of hMG doses (75 IU FSH and 75 IU LH) was 37.4 ± 16.5 and the mean duration of stimulation was 8.9 ± 1.6 days. A total of 827 oocytes (oocyte per cycle: 11.0 ± 5.2) was retrieved. Four hundred ninety-four oocytes were retrieved from patients whose partner did not suffer from any diagnosed infertility (female infertility only). As described in Englert et al. (6), the oocytes were cultured individually in petri dishes containing modified Earles' Balanced Salt Solution drops and semen samples were prepared at the day of oocyte retrieval (3). The cumulus maturity was observed under an inverted light microscope at 100 to 200x magnification and were classified as mature, overmature, or immature according to the cumulus expansion. The insemination took place after 3 to 6 hours of oocyte incubation according to the cumulus maturity assessment. Sixteen to 20 hours after insemination, the cumulus oophorus was removed mechanically by passing the oocytes through a small pasteur pipette. Fertilization was determined by the presence of two pronuclei, and the ZP thickness was measured as described in Bertrand et al. (3). The measurements were made under an inverted light microscope equipped with Nomarski optics (Olympus MT2, Tokyo, Japan) at 400x magnification with a calibrated eyepiece graticule. A total of 827 oocytes was analyzed; the fertilization rate reached 53% with a polyspermic rate of 2.6%. In women of couples suffering from female infertility only, the fertilization rate reached 65% (494 oocytes) with a polyspermic rate of 3%. As a control to assess the influence of the fertilization process on the ZP thickness, ZP of 75 additional oocytes were measured before and 16 to 20 hours after intracytoplasmic single spermatozoon injection (ICSI). Vol. 66, No.3, September 1996
Twenty-four hours after fertilization assessment, the embryo quality was evaluated according to Puissant et al. (7). Sixty-eight transfers occurred, 177 embryos were replaced, and 23 patients were pregnant (35%). Statistical Analyses
Comparison between the ZP thickness of fertilized, polyspermic, and unfertilized oocytes was tested by one-way analysis of variance (ANOVA) and the multiple comparison test of Fisher (8). For patients having followed several cycles, the mean ZP thickness of oocytes retrieved during the different cycles were compared (ANOVA + multiple comparison test). The relationships between the ZP thickness and the age of the women, the duration of stimulation, the cumulus maturity, the number of retrieved 00cytes, the number ofhMG doses administered to the patients, the maximum E2 level, and the follicular volume were tested using linear regression analysis (least square method). Semilogarithmic, bilogarithmic, and inverse transformations of the data were performed to test the biologic relevant nonlinear regressions (exponential, power, and hyperbolic regressions, respectively) (8). Presented regression models are those significant (a = 0.05) and showing the best fit (highest determination coefficient). All data are expressed as means ± SD. RESULTS
The mean ZP thickness of fertilized (n = 304), polyspermic (n = 16), and unfertilized (n = 174) 00cytes of patients suffering from female infertility only were compared by one-way ANOVA and a multiple comparison test. The mean ZP thickness of fertilized (16.4 ± 3.1 p,m) and polyspermic oocytes (15.1 ± 2.4 p,m) were significantly smaller than the mean ZP thickness of unfertilized oocytes (18.9 ± 3.8 p,m) (P = 2.5 x 10-6 ). The difference between the mean ZP thickness of fertilized and polyspermic oocytes was barely significant (P = 0.1). Among all the selected patients, 10 underwent several cycles and the mean ZP thickness during their different cycles were compared by a multiple comparison test. In all but two cases, the mean ZP thickness of a patient was not significantly different from one cycle to the other(s). When a significant difference did appear, it was two to three times smaller than the interindividual variation (maximal intraindividual difference: 3.3 p,m versus maximal interindividual difference: 10.7 p,m). Comparisons also were made between the mean ZP thickness and available clinical parameters susceptible to influence the ZP size. The age of the Bertrand et al. Zona pellucida thickness
409
Table 1 Results of the Linear Regressions Between the Mean ZP Thickness and the Clinical Personal Parameters of the Patients Parameters
Determination coefficient
Age of the patients Stimulation duration No. of hMG doses Follicular volume Cumulus maturity No. of retrived oocytes Maximum E2 level
0.004 0.006 0.15 0.01 0.02 0.04 0.09
p
Slope
NSt NS 2.6
0.008 NS NS 0.009
+
+ +
+ + + + + ++++ ++ + + +++ + ++ + 1$·+ + + + + + ....... + +++ +*.t+ ++ J++ + t+ + + + + + + +
-
2
*
*
+ +
0 11
13
15
17 19 ZP thickness (pm)
21
23
25
Figure 1 Linear regression between maximum E2 level and mean ZP thickness. n = 75 cycles; y= -0.13x + 4.93; R2 = 0.09; SE (slope) = :<::0.05; SE (intercept) = :<::0.87. 410
++
40 o 30
+
+++~
+++ ++ + "" ... + '*'Iq; +
20 10
13
+
+ ++
++~ ++
+
...~
+
15
+ + +
17 19 ZP thickness C!un)
21
23
25
Figure 2 Linear regression between number ofhMG doses and mean ZP thickness. n = 75 cycles; y = 2.65x - 9.06; R2 = 0.15; SE (slope) = :<::0.76; SE (intercept) = :<::13.43.
A significant difference of the ZP thickness occurs between fertilized and unfertilized oocytes, the former ones having a thinner ZP thickness than the other ones. This was shown already on a limited sample in an earlier study (3). Moreover, in the pres-
S3
+ +
-0.13
DISCUSSION
1;a
+
+
70 ~ 60
11
NS
women, the duration of stimulation, the cumulus maturity, the number of retrieved oocytes, the number of hMG doses administered to the patients, the maximum E2 level, and the follicular volume were taken into account. The results are reported in Table 1. A significant decreasing relationship was found between the ZP thickness and the maximum E2 level and an increasing one was found with the hMG dose (Figs. 1 and 2). The relationships with the other parameters appeared to be nonsignificant. The ZP of 75 additional oocytes were measured before (17.48 ± 2.48) and after (17.5 ± 2.43) ICSI to assess the influence of the fertilization process on the zona pellucida thickness. The fertilization rate reached 71%. Statistical analysis (ANOVA) revealed no significant difference.
i4
...,
+
." ~50
A
* n = 75 cycles. t NS, not significant.
5
90 80
Bertrand et al. Zona pellucida thickness
ent study, the mean ZP thickness of polyspermic 00cytes also has been compared with the one of other types of oocytes. The ZP thickness of more than two pronuclear oocytes presented the lowest mean ZP thickness of the 494 measured oocytes. However, the difference between the ZP thickness of oocytes fertilized by one spermatozoon or by several spermatozoa is barely significant. It looks as though there is an optimal size of ZP thickness beyond which fertilization is difficult and below which the block to polyspermia is not efficient enough. Hence, ZP thickness seems to be a key factor in IVF-ET process. Furthermore Cohen et al. (9) and Janssens et al. (Janssens R, Carle M, De Clerck E, Henderix P, Laurier K, Nagy R, et aI., abstract) have shown that ZP thickness also plays an important role in embryo implantation process. According to our results, ZP thickness ofICSI oocytes is not affected by fertilization. The ZP is thus a characteristic of oocytes that exists before fertilization (75 ICSI oocytes analyzed before and 16 to 20 hours after ICSI: no significant difference). Wassarman et al. (10) have shown that, during fertilization, the glycoproteins of the ZP (ZP2 and ZP3) undergo a protease and a glycosidase reaction, respectively, leading to a change into ZP2 f and ZP3 f • Cohen et al. (9) have shown that a thinning procedure occurs from 54 hours after fertilization. It thus can be assumed that ZP is affected qualitatively during fertilization, but thinning of ZP thickness is a postfertilization process that occurs only a few days later. The present study focuses particularly on parameters susceptible to influence the ZP thickness. In a first step, the mean ZP thickness of same patients having followed several cycles was compared. In nearly all cases, no significant difference was observed from one cycle to the other(s) of a same patient. In a previous study (3), it has been shown that IVF-ET patients can be gathered in different groups according to their significantly different mean ZP Fertility and Sterility®
thickness. Those groups of patients with increasing mean ZP thickness presented a decreasing fertilization rate. According to the available data, ZP thickness basically is an individual feature. This feature nevertheless may be modified slightly by clinical parameters specific to hormonal cycles. Among the relationships between ZP thickness and the studied clinical parameters, the relationships with the E2 level in the peripheral plasma (inverse linear relationship) and with the number of hMG doses (direct linear relationship) are significant. It has been shown that E2 plays an important role in oocyte cytoplasmic maturation (11). Several studies have attempted to establish correlations between steroid concentrations and the quality of follicles or oocyte maturity (12-17). In stimulated cycles, a correlation between follicular steroids, namely E 2 , and oocyte fertilizability in vitro has been shown (12, 13). Indeed, oocytes giving rise to fertilization and pregnancy come from the most estrogenic follicles (14, 15). In the present study, fertilizable oocytes presented a small ZP thickness and are those exposed to high E2 levels, as E2 in peripheral plasma derive almost solely from the actively growing follicles (16, 17). So, good stimulation characteristics are reflected by the E2 level, which seems to be indispensable for oocyte maturation. The latter corresponds also to an optimal ZP thickness, which influences the fertilization rate. The relationship with the number of hMG doses agreed with the previous idea that "good responder" patients require few hMG doses in order to reach a high E2 level. This level seems to be a prerequisite for oocytes having a small ZP thickness and thus presenting a high fertilization rate. This suggests that the characteristics of a good stimulation can be reflected by the ZP thickness. It would be worth investigating linear relationships between ZP thickness and E2 and hMG of same patients having followed several cycles. However, patients usually follow too few cycles to provide the hormonal stimulation data required for statistical analysis. In conclusion, it can be stated that ZP thickness is basically an individual feature which influences the fertilization rate. Zona pellucida thickness may nevertheless be slightly influenced by the E2 level, and it reflects the quality of hormonal stimulation.
Vo!' 66, No.3, September 1996
Acknowledgments. We thank Francis Cantraine, M.Sc, Ph.D., head of the Department of Computer Science (Free University of Brussels) for statistical revision of the manuscript.
REFERENCES 1. Dietl JA, Rauth G. Molecular aspects of mammalian fertilization. Hum Reprod 1989;4:869-75. 2. Dean J. Biology of mammalian fertilization: role of the zona pellucida. J Clin Invest 1992;89:1055-9. 3. Bertrand E, Van den bergh M, Englert Y. Does zona pellucida thickness influence the fertilization rate? Hum Reprod 1995; 10:1189-93. 4. Wassarman P. Regulation of mammalian fertilization by zona pellucida glycoproteins. J Reprod Fertil 1990;42:79-87. 5. Arnot AM, Vandekerckhove P, DeBono MA, Rutherford A. Follicular volume and number during in vitro fertilization: association with oocyte developmental capacity and pregnancy rate. Hum Reprod 1995; 10:256-61. 6. Englert Y, Van den Bergh M, Rodesch C, Bertrand E, Biramane J, Legreve A. Comparative auto-controlled study between swim-up and Percoll preparation of fresh semen samples for in-vitro fertilization. Hum Reprod 1992; 7:399-402. 7. Puissant F, Van Rysselberge M, Barlow P, Deweze J, Leroy F. Embryo scoring as a prognostic tool in IVF-ET treatment. Hum Reprod 1987;2:705-8. 8. Zar JK, editor. Biostatistical analysis. New Jersey: PrenticeHall Inc, 1984. 9. Cohen J, Alikani M, Trowbridge J, Rosenwaks Z. Implantation enhancement by selective assisted hatching using zona drilling of human embryos with poor prognosis. Hum Reprod 1992;7:685-91. 10. Wassarman P. Mouse gamete adhesion molecules. BioI Reprod 1992;46:186-91. 11. Thibault C. Are follicular maturation and oocyte maturation independent processes? J Reprod FertiI1977;51:1-15. 12. Botero-Ruiz W, Laufer N, DeCherney AH, Polan ML, Haseltime FP, Behrman HR. The relationship between follicular fluid steroid concentration and successful fertilization of human oocytes in vitro. Fertil Steril 1984;41:820-6. 13. Kreiner D, Liu H-C, Itskovitz J, Veeck L, Rosenwaks Z. Follicular fluid estradiol and progesterone are markers ofpreovulatory oocyte quality. Fertil Steril 1987;48:991-4. 14. Carson R, Trounson A, Findlay J. Successful fertilization of human oocytes in vitro: concentration of estradiol-17,B, progesterone and androstenedione in the antral fluid of donor follicles. J Clin Endocrinol Metab 1982;55:798-800. 15. Wramsby H, Kullander S, Liedholm P, Rannevik G, Sundstrom P, Thorell J. The success rate of in vitro fertilization of human oocytes in relation to the concentrations of different hormones in follicular fluid and peripheral plasma. Fertil Steril 1981;36:448-54. 16. McNatty K, Hunter W, McNeilly A, Sawers R. Changes in the concentration of pituitary and steroid hormones in the follicular fluid of human graafian follicles throughout the menstrual cycle. J Endocrinol 1975;64:555-71. 17. McNatty K, Baird D, Bolton A, Chambers P, Corker C, McLean H. Concentration of oestrogens and androgens in human ovarian venous plasma and follicular fluid throughout the menstrual cycle. J Endocrinol 1976; 71:77 -85.
Bertrand et aI. Zona pellucida thickness
411
Vol. 66, No.3, September 1996
Printed on acid-free paper in U. S. A
Copyright c 1996 American Society for Reproductive Medicine
Clinical parameters influencing human zona pellucida thickness*
Evelyne Bertrand, M.Sc.t Marc Van den Bergh, M.T. Yvon Englert, M.D., Ph.D. Department of Obstetric and Gynecology, Fertility Unit, Free University of Brussels, Erasme Hospital, Brussels, Belgium
Objectives: To assess which clinical parameters influence human oocyte zona pellucida (ZP) thickness. Patients: Sixty-five couples undergoing 75 IVF-ET cycles. Main Outcome Measure: Zona pellucida thickness of 827 oocytes measured 16 to 20 hours after in vitro insemination under inverted light microscope. Results: Zona pellucida thickness was 18.9 ± 3.8 p,m (mean ± SD) for unfertilized, 16.4 ± 3.1 p,m for fertilized, and 15.1 ± 2.4 p,m for polyspermic oocytes (significantly different). Among our patients, a few underwent two (or even three) IVF-ET cycles, and the mean ZP thickness was, in most cases, not significantly different from one cycle to the other(s). Regression analyses were calculated between ZP thickness and available clinical parameters, i.e., the age of the women, the duration of stimulation, the cumulus maturity, the number of retrieved oocytes, the number ofhMG doses, the maximum E 2 level, and the follicular volume. A significant linear decreasing relationship exists between the mean ZP thickness of each patient and the maximum E2 level and an increasing one with the hMG dose. Relationships with the other parameters appeared to be nonsignificant. Conclusion: The ZP thickness is basically an individual feature that influences the fertilization rate. Nevertheless, it may be influenced slightly by the hormonal treatment during stimulation. Fertil Steril® 1996; 66:408-11 Key Words: Fertilization rate, human oocytes, IVF-ET, zona pellucida thickness, hormonal stimulation
Gamete-specific glycoproteins compose the relatively thick acellular glycoprotein coat, the zona pellucida (ZP), and participate in various essential functions of the fertilization process (1, 2). Its thickness varies between mammalian species and also between individuals of a single species. The ZP thickness seems to be correlated directly with protein content (3,4). It generally is considered that fertilization rate in IVF-ET is closely dependent on sperm characteristics. Nevertheless, a previous study on human oocytes fertilized in vitro has shown that ZP thickness has an influence on the fertilization rate because fertilized oocytes have, on average, a significantly thinner ZP than unfertilized oocytes (3). Received August 3,1995; revised and accepted April 11, 1996. * Supported by the Fonds National de Ie Recherche Scientifique, Brussels, Belgium. t Reprint requests: Evelyne Bertrand, M.Sc., Department of Obstetric and Gynecology, Fertility Unit, Erasme Hospital, 808 Route de Lennik, 1070 Brussels, Belgium (FAX.: 32-25556672). 408
Bertrand et al. Zona pellucida thickness
The failure of IVF-ET may be linked to a general oocyte dysfunction, the ZP thickness being only a marker of it. This dysfunction could be intrinsic to the oocyte, to the patient, or linked to clinical characteristics such as hormonal stimulation. The aim of the present study was to assess, in an IVF-ET program, which available clinical parameters could intervene in the ZP thickness. MATERIALS AND METHODS Patient Data
Zona pellucida thickness was measured on oocytes collected during 75 IVF-ET cycles performed in 65 couples. Among those patients, 44 couples suffering only from female infertilities were selected to assess the difference between unfertilized, fertilized (two pronuclei), and polyspermic oocytes (more than two pronuclei). The women ranged in age from 23 to 42 years (34.2 Fertility and Sterility®
± 4.0 years; mean ± SD). The duration of infertility was 6.5 ± 4.1 years. Within the 65 couples, 8 underwent two cycles and 2 underwent three cycles. Treatment Protocol
Mter classical follicular stimulation using hMG (Humegon; Organon, Oss, The Netherlands and Pergonal; Serono, Milan, Italy) under LH-releasing hormone analogue (buserelin acetate, Suprefact; Hoechst Inc., Frankfurt, Germany), oocytes were picked up under transvaginal ultrasonic guidance, 36 hours after ovulation induction by 104 IU hCG (Profasi; Serono and Pregnyl; Organon). Follicles were aspirated and the volume of follicular fluid obtained from each aspirated follicle was measured as described by Arnot et al. (5). The mean follicular volume was 3.7 ± 2.0 mL. The mean maximum E2 level in the peripheral plasma was evaluated by RIAs and reached 2.6 ± 1.1 ng/mL (conversion factor to SI unit, 3.671). The mean number of hMG doses (75 IU FSH and 75 IU LH) was 37.4 ± 16.5 and the mean duration of stimulation was 8.9 ± 1.6 days. A total of 827 oocytes (oocyte per cycle: 11.0 ± 5.2) was retrieved. Four hundred ninety-four oocytes were retrieved from patients whose partner did not suffer from any diagnosed infertility (female infertility only). As described in Englert et al. (6), the oocytes were cultured individually in petri dishes containing modified Earles' Balanced Salt Solution drops and semen samples were prepared at the day of oocyte retrieval (3). The cumulus maturity was observed under an inverted light microscope at 100 to 200x magnification and were classified as mature, overmature, or immature according to the cumulus expansion. The insemination took place after 3 to 6 hours of oocyte incubation according to the cumulus maturity assessment. Sixteen to 20 hours after insemination, the cumulus oophorus was removed mechanically by passing the oocytes through a small pasteur pipette. Fertilization was determined by the presence of two pronuclei, and the ZP thickness was measured as described in Bertrand et al. (3). The measurements were made under an inverted light microscope equipped with Nomarski optics (Olympus MT2, Tokyo, Japan) at 400x magnification with a calibrated eyepiece graticule. A total of 827 oocytes was analyzed; the fertilization rate reached 53% with a polyspermic rate of 2.6%. In women of couples suffering from female infertility only, the fertilization rate reached 65% (494 oocytes) with a polyspermic rate of 3%. As a control to assess the influence of the fertilization process on the ZP thickness, ZP of 75 additional oocytes were measured before and 16 to 20 hours after intracytoplasmic single spermatozoon injection (ICSI). Vol. 66, No.3, September 1996
Twenty-four hours after fertilization assessment, the embryo quality was evaluated according to Puissant et al. (7). Sixty-eight transfers occurred, 177 embryos were replaced, and 23 patients were pregnant (35%). Statistical Analyses
Comparison between the ZP thickness of fertilized, polyspermic, and unfertilized oocytes was tested by one-way analysis of variance (ANOVA) and the multiple comparison test of Fisher (8). For patients having followed several cycles, the mean ZP thickness of oocytes retrieved during the different cycles were compared (ANOVA + multiple comparison test). The relationships between the ZP thickness and the age of the women, the duration of stimulation, the cumulus maturity, the number of retrieved 00cytes, the number ofhMG doses administered to the patients, the maximum E2 level, and the follicular volume were tested using linear regression analysis (least square method). Semilogarithmic, bilogarithmic, and inverse transformations of the data were performed to test the biologic relevant nonlinear regressions (exponential, power, and hyperbolic regressions, respectively) (8). Presented regression models are those significant (a = 0.05) and showing the best fit (highest determination coefficient). All data are expressed as means ± SD. RESULTS
The mean ZP thickness of fertilized (n = 304), polyspermic (n = 16), and unfertilized (n = 174) 00cytes of patients suffering from female infertility only were compared by one-way ANOVA and a multiple comparison test. The mean ZP thickness of fertilized (16.4 ± 3.1 p,m) and polyspermic oocytes (15.1 ± 2.4 p,m) were significantly smaller than the mean ZP thickness of unfertilized oocytes (18.9 ± 3.8 p,m) (P = 2.5 x 10-6 ). The difference between the mean ZP thickness of fertilized and polyspermic oocytes was barely significant (P = 0.1). Among all the selected patients, 10 underwent several cycles and the mean ZP thickness during their different cycles were compared by a multiple comparison test. In all but two cases, the mean ZP thickness of a patient was not significantly different from one cycle to the other(s). When a significant difference did appear, it was two to three times smaller than the interindividual variation (maximal intraindividual difference: 3.3 p,m versus maximal interindividual difference: 10.7 p,m). Comparisons also were made between the mean ZP thickness and available clinical parameters susceptible to influence the ZP size. The age of the Bertrand et al. Zona pellucida thickness
409
Table 1 Results of the Linear Regressions Between the Mean ZP Thickness and the Clinical Personal Parameters of the Patients Parameters
Determination coefficient
Age of the patients Stimulation duration No. of hMG doses Follicular volume Cumulus maturity No. of retrived oocytes Maximum E2 level
0.004 0.006 0.15 0.01 0.02 0.04 0.09
p
Slope
NSt NS 2.6
0.008 NS NS 0.009
+
+ +
+ + + + + ++++ ++ + + +++ + ++ + 1$·+ + + + + + ....... + +++ +*.t+ ++ J++ + t+ + + + + + + +
-
2
*
*
+ +
0 11
13
15
17 19 ZP thickness (pm)
21
23
25
Figure 1 Linear regression between maximum E2 level and mean ZP thickness. n = 75 cycles; y= -0.13x + 4.93; R2 = 0.09; SE (slope) = :<::0.05; SE (intercept) = :<::0.87. 410
++
40 o 30
+
+++~
+++ ++ + "" ... + '*'Iq; +
20 10
13
+
+ ++
++~ ++
+
...~
+
15
+ + +
17 19 ZP thickness C!un)
21
23
25
Figure 2 Linear regression between number ofhMG doses and mean ZP thickness. n = 75 cycles; y = 2.65x - 9.06; R2 = 0.15; SE (slope) = :<::0.76; SE (intercept) = :<::13.43.
A significant difference of the ZP thickness occurs between fertilized and unfertilized oocytes, the former ones having a thinner ZP thickness than the other ones. This was shown already on a limited sample in an earlier study (3). Moreover, in the pres-
S3
+ +
-0.13
DISCUSSION
1;a
+
+
70 ~ 60
11
NS
women, the duration of stimulation, the cumulus maturity, the number of retrieved oocytes, the number of hMG doses administered to the patients, the maximum E2 level, and the follicular volume were taken into account. The results are reported in Table 1. A significant decreasing relationship was found between the ZP thickness and the maximum E2 level and an increasing one was found with the hMG dose (Figs. 1 and 2). The relationships with the other parameters appeared to be nonsignificant. The ZP of 75 additional oocytes were measured before (17.48 ± 2.48) and after (17.5 ± 2.43) ICSI to assess the influence of the fertilization process on the zona pellucida thickness. The fertilization rate reached 71%. Statistical analysis (ANOVA) revealed no significant difference.
i4
...,
+
." ~50
A
* n = 75 cycles. t NS, not significant.
5
90 80
Bertrand et al. Zona pellucida thickness
ent study, the mean ZP thickness of polyspermic 00cytes also has been compared with the one of other types of oocytes. The ZP thickness of more than two pronuclear oocytes presented the lowest mean ZP thickness of the 494 measured oocytes. However, the difference between the ZP thickness of oocytes fertilized by one spermatozoon or by several spermatozoa is barely significant. It looks as though there is an optimal size of ZP thickness beyond which fertilization is difficult and below which the block to polyspermia is not efficient enough. Hence, ZP thickness seems to be a key factor in IVF-ET process. Furthermore Cohen et al. (9) and Janssens et al. (Janssens R, Carle M, De Clerck E, Henderix P, Laurier K, Nagy R, et aI., abstract) have shown that ZP thickness also plays an important role in embryo implantation process. According to our results, ZP thickness ofICSI oocytes is not affected by fertilization. The ZP is thus a characteristic of oocytes that exists before fertilization (75 ICSI oocytes analyzed before and 16 to 20 hours after ICSI: no significant difference). Wassarman et al. (10) have shown that, during fertilization, the glycoproteins of the ZP (ZP2 and ZP3) undergo a protease and a glycosidase reaction, respectively, leading to a change into ZP2 f and ZP3 f • Cohen et al. (9) have shown that a thinning procedure occurs from 54 hours after fertilization. It thus can be assumed that ZP is affected qualitatively during fertilization, but thinning of ZP thickness is a postfertilization process that occurs only a few days later. The present study focuses particularly on parameters susceptible to influence the ZP thickness. In a first step, the mean ZP thickness of same patients having followed several cycles was compared. In nearly all cases, no significant difference was observed from one cycle to the other(s) of a same patient. In a previous study (3), it has been shown that IVF-ET patients can be gathered in different groups according to their significantly different mean ZP Fertility and Sterility®
thickness. Those groups of patients with increasing mean ZP thickness presented a decreasing fertilization rate. According to the available data, ZP thickness basically is an individual feature. This feature nevertheless may be modified slightly by clinical parameters specific to hormonal cycles. Among the relationships between ZP thickness and the studied clinical parameters, the relationships with the E2 level in the peripheral plasma (inverse linear relationship) and with the number of hMG doses (direct linear relationship) are significant. It has been shown that E2 plays an important role in oocyte cytoplasmic maturation (11). Several studies have attempted to establish correlations between steroid concentrations and the quality of follicles or oocyte maturity (12-17). In stimulated cycles, a correlation between follicular steroids, namely E 2 , and oocyte fertilizability in vitro has been shown (12, 13). Indeed, oocytes giving rise to fertilization and pregnancy come from the most estrogenic follicles (14, 15). In the present study, fertilizable oocytes presented a small ZP thickness and are those exposed to high E2 levels, as E2 in peripheral plasma derive almost solely from the actively growing follicles (16, 17). So, good stimulation characteristics are reflected by the E2 level, which seems to be indispensable for oocyte maturation. The latter corresponds also to an optimal ZP thickness, which influences the fertilization rate. The relationship with the number of hMG doses agreed with the previous idea that "good responder" patients require few hMG doses in order to reach a high E2 level. This level seems to be a prerequisite for oocytes having a small ZP thickness and thus presenting a high fertilization rate. This suggests that the characteristics of a good stimulation can be reflected by the ZP thickness. It would be worth investigating linear relationships between ZP thickness and E2 and hMG of same patients having followed several cycles. However, patients usually follow too few cycles to provide the hormonal stimulation data required for statistical analysis. In conclusion, it can be stated that ZP thickness is basically an individual feature which influences the fertilization rate. Zona pellucida thickness may nevertheless be slightly influenced by the E2 level, and it reflects the quality of hormonal stimulation.
Vo!' 66, No.3, September 1996
Acknowledgments. We thank Francis Cantraine, M.Sc, Ph.D., head of the Department of Computer Science (Free University of Brussels) for statistical revision of the manuscript.
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