FERTILITY AND STERILITY
Vol. 67, No. 1, January 1992
Printed on acid-free paper in U.S.A.
Copyright 0 1992 The American Fertility Society
Increased fertilization and pregnancy rate in polypronuclear fertilization cycles in in vitro fertilization-embryo transfer
Abraham Golan, M.D.* Hannah Nachum Arie Herman, M.D.
Raphael Ron-El, M.D. Yigal Soffer, M.D. Eliahu Caspi, M.D.
Department of Obstetrics and Gynecology, Assaf Harofeh Medical Center, Zerifin, Israel
Objective: To compare fertilization and pregnancy rates between cycles with polypronuclear fertilizations and cycles with normal fertilizations. Design: In vitro fertilization-embryo transfer (IVF-ET) cycles in which oocytes were retrieved were divided into two groups according to the nature of fertilization. Patients: All patients were participants of our IVF-ET program. Results: A significantly higher fertilization rate was found in the polypronuclear fertilization cycles (61% versus 36.6%) and also an improved pregnancy rate (47.5% versus 19.6%) and per embryo transfers (53% versus 28.8%). The vast majority ofpolypronuclear fertilizations occurred in mature oocytes. Conclusion: We believe that the increased receptability of the oocytes improves fertilization and conception rates. The polypronuclear fertilization is an extreme expression of such improved receptibility and should be considered as an encouraging sign for conception. Fertil Steril 1992;57:139-42
The phenomenon of polypronuclear fertilization is accepted as a common, though undesirable, outcome of in vitro fertilization (IVF). Various authors reported its occurrence in IVF programs with an incidence of2% to 10%. 1- 3 The impression that fertilization and conception rates are exceptionally high in treatment cycles with such polypronuclear fertilization have stimulated us to investigate this phenomenon in our program.
MATERIALS AND METHODS Out of 832 consecutive IVF treatment cycles performed between April 1986 and March 1990, 646 successful oocyte retrievals were performed. Four different stimulation protocols were used:
Received December 21, 1990; revised and accepted September 4, 1991. * Reprint requests: Abraham Golan, M.D., Obstetrics and Gynecology, Assaf Harofeh Medical Center, Zerifin 70300, Israel. Vol. 57, No. 1, January 1992
1. Human merwpausealgonadotropin (hMG) (135 cycles): hMG (Pergonal; Teva, Petah Tikva, Israel) was started on day 3 at three ampules per day. 2. Early folicular D-Trp6 fhMG (213 cycles): An intramuscular injection of 3.2 mg of D-Trp6 luteinizing hormone-releasing hormone (LH-RH) (Decapeptyl; Ferring, Malmo, Sweden) was administered between days 1 and 3 of the menstrual cycle . When ovarian suppression was confirmed (estradiol [E 2 ] ~30 pgfmL and no follicle cyst on ultrasound) hMG treatment (3 ampules daily) was commenced (usually on day 18). 3. MidlutealD-Trp6 fhMG (227 cycles): The same protocol as in group 2 starting in the midluteal phase (usually day 22 to 24). 4. Ultrashort D-Trp6 fhMG (71 cycles): hMG was started as described in group 1. On day 2, a daily dose of 0.5 mg of D-Trp 6 LH-RH was administered intramuscularly for 3 consecutive days (days 2 to 4). In all our groups hMG was administered at three ampules per day and individually adjusted. Ovarian stimulation was monitored by daily E 2 , progesterone, Golan et al.
Polypronuclei indicate more IVF conceptions
139
and LH serum levels, and by ovarian ultrasonography. Human chorionic gonadotropin (10,000 IU, Chorigon; Teva) was given when the leading follicle was ~18 mm in diameter. Oocytes were retrieved vaginally under ultrasonographic control in the majority of the cases (95%), and the rest were retrieved laparoscopically. Up to four embryos were replaced and the rest cryopreserved. The suction pressure at oocyte retrieval was 120 to 160 mmHg, rarely increasing to 200 mmHg. The oocytes were inseminated 1 to 4 hours later using a semen concentration of 150,000 to 350,000 sperm/mL culture medium in center well plates. Fertilization was determined 18 to 22 hours later after mechanical removal of cumulus cells and the observation of two pronuclei. The X2 test with a continuity correction was used for statistical analysis.
RESULTS Out of the 646 cycles with oocytes retrievals, polypronuclear fertilization occurred in 40 cycles (in 36 patients). These cycles yielded 315 oocytes and 192 embryos out of which polypronuclear fertilization was found in 56. Among all the cycles with retrieved oocytes (646), the incidence of cycles with polypronuclear fertilization was 6.2%. The 56 polypronuclear embryos were 2.8% of the total of 1,987 embryos obtained. In the hMG group, the polypronuclear fertilization occurred in 45% of the cycles and was 2.1% of the total number of embryos. In the early follicular DTrp6/hMG group, the polypronuclear fertilization occurred in 3.8% of the cycles and was 1.3% of the total number of embryos. In the midluteal D-Trp6I hMG group, the polypronuclear fertilization occurred in 8.4% of the cycles and was 4% of the total number of embryos. In the ultrashort D-Trp6/hMG group, the polypronuclear fertilization occurred in 9.9% of the cycles and was 4.7% of the total number of embryos (Table 1). The rates of polypronuclear Table 1
fertilization in the various treatment protocol groups did not significantly differ (X 2 test, P = 0.1). In 12 cycles (one-fifth of the 40 polypronuclear fertilization cycles), there was more than one polypronuclear fertilization. Two triploid embryos were observed in 7 of the cycles, 4 triploid embryos in 2, a triploid and a tetraploid in 1, a triploid and a pentaploid in 1, and 2 hexaploids in 1 cycle (totaling 28 polypronuclear embryos in 12 cycles). The majority of the polypronuclear fertilizations occurred in mature oocytes. Of the 315 oocytes, 289 (92%) were mature and only 26 immature. Polypronuclear fertilization occurred in mature oocytes in 36 cycles and in immature oocytes in 6, (2 of the cycles comprised of both mature and immature oocytes). The average preinsemination oocyte incubation time in the polypronuclear group (2.34 hours) did not differ significantly from that of the normal fertilization group (2.66 hours) (P = 0.8). The vast majority of the patients with polypronuclear fertilization (33 of the 36 patients) suffered from unexplained infertility. Male factor was the cause of the infertility in two of the patients, and only one patient had a tubal cause for her infertility. In 36 of the 40 cycles, at least one embryo was replaced. The polypronuclear embryos were not replaced. Of a total of 221 patients aged ::::;;30 years, 17 had polypronuclear fertilizations. Twenty-eight cases of polypronuclear fertilization occurred among a total of 425 patients > 30 years of age. The difference was not statistically significant (P = 0. 7). Fertilization rate differed significantly between the polypronuclear fertilization cycles and the rest. Of the 315 oocytes, 192 fertilizations occurred (a fertilization rate of 61%) as compared with a fertilization rate of 36.6% in the normal cycles (of 4,442 oocytes 1,625 fertilizations) (P < 0.00001, X2 test) (Table 2). Pregnancy rate in the study group was also significantly higher. In the 40 polypronuclear fertiliza-
Incidence of Polypronuclear Fertilization Polypronuclear fertilization
Treatment protocol HMG Early follicular D-Trp6/hMG Midluteal D-Trp6/hMG Ultrashort D-Trp6 /hMG Total
140
Golan et al.
No. cycles
No. embryos
No. cycles
Percentage of cycles
No. embryos
Percentage of embryos
135 213 227
327 699 749 212 1,987
6 8 19 7 40
4.45 3.75 8.37 9.86 6.19
7 9 30 10 56
2.1 1.3 4.0 4.7 2.8
71
646
Polypronuclei indicate more IVF conceptions
Fertility and Sterility
Table 2
Fertilization and Pregnancy Rates Cycles with oocyte retrieval
No. of oocytes
No. of embryos
Fertilization rate
Clinical pregnancies
%
Polypronuclear fertilization Normal fertilization
Pregnancy rate/oocyte retrieval
Pregnancy rate/embryo transfer
%
%
40
315
192
60.9"
19
47.5b
sa·
604
4,442
1,625
36.6"
119
19.6b
28.8.
P < 0.00001. p < 0.0001. c p < 0.006. a
b
tion cycles, 19 clinical pregnancies occurred, with a pregnancy rate of 47.5% per cycle resulting in oocytes, compared with 119 pregnancies of the other 606 cycles, with a rate of 19.64% (P < 0.0001, X2 test) (Table 2). The pregnancy rate per embryo transfer in the polypronuclear group was 19 of 36 (53%) as compared with 119 of 444 (28.8%) in the normal fertilization cycles (P < 0.006). Three more patients of the polypronuclear group conceived in further IVF cycles. Among the 12 cycles with multiple polypronuclear fertilizations, 7 ended in pregnancy (58.5%) and an additional pregnancy occurred in a consecutive cycle. DISCUSSION
The most interesting finding in this study is the fact that fertilization and conception rates were significantly higher in cycles with polypronuclear fertilizations. This observation has been recently reported. 4•5 Increased fertilizing capacity of the sperm or improved oocyte receptibility could be the explanation. Polyspermy is believed to be the major factor involved in the occurrence ofpolypronuclear fertilization. Few explanations have been suggested for the increased incidence of polyspermy in vitro: (1) early insemination, (2) increased number of sperms surrounding the oocyte, and (3) delayed insemination. Sathananthan and Trounson6 observed a delayed release of cortical granules in polyspermic oocytes indicating delayed cortical maturation of the oocytes, eventually being the cause of polyspermy. In normal monospermic fertilization, cortical granules are released into the perivitaline space, decondense and swell, then interact with the inner layer of the zona pellucida and initiate a change in this area that prevents any further penetration by sperm. These authors suggested that polyspermy could be prevented by incubation for a few hours (up to 6 hours) Vol. 57, No. 1, January 1992
before insemination, thus improving cortical maturation.7 The large number of motile sperm surrounding the oocyte in IVF as compared with the smaller number in vivo has also been suggested as a possible etiological factor. Wolf et al. 8 clearly demonstrated that an increase in sperm concentration is directly related to an increase in the incidence of polyspermy. Plachot et al. 2 demonstrated a lower rate of polyspermy when sperm quality was reduced. The limit of 50,000 motile sperm per 1 mL of culture medium was suggested by Sathananthan and Trounson6 in an effort to reduce the incidence of polyspermia. Diamond et al., 3 however, did not find any effect of different sperm concentrations (125,000 to 500,000 per oocyte). Unlike previous reports by Sathananthan and Trounson6 and Trounson et al. 7 and Van der Ven et al., 9 Rudak et al. 1 found polypronuclear fertilization to predominantly occur in mature oocytes. They reported that a third of their polypronuclear fertilization occurred in postmature oocytes and none in immature oocytes. This seems to be in close agreement with our finding that polypronuclear fertilization occurs in mature rather than in immature oocytes. Plachot et al. 2 found that polyspermy is reduced when the preinsemination incubation period is short (
Vol. 67, No. 1, January 1992
Printed on acid-free paper in U.S.A.
Copyright 0 1992 The American Fertility Society
Increased fertilization and pregnancy rate in polypronuclear fertilization cycles in in vitro fertilization-embryo transfer
Abraham Golan, M.D.* Hannah Nachum Arie Herman, M.D.
Raphael Ron-El, M.D. Yigal Soffer, M.D. Eliahu Caspi, M.D.
Department of Obstetrics and Gynecology, Assaf Harofeh Medical Center, Zerifin, Israel
Objective: To compare fertilization and pregnancy rates between cycles with polypronuclear fertilizations and cycles with normal fertilizations. Design: In vitro fertilization-embryo transfer (IVF-ET) cycles in which oocytes were retrieved were divided into two groups according to the nature of fertilization. Patients: All patients were participants of our IVF-ET program. Results: A significantly higher fertilization rate was found in the polypronuclear fertilization cycles (61% versus 36.6%) and also an improved pregnancy rate (47.5% versus 19.6%) and per embryo transfers (53% versus 28.8%). The vast majority ofpolypronuclear fertilizations occurred in mature oocytes. Conclusion: We believe that the increased receptability of the oocytes improves fertilization and conception rates. The polypronuclear fertilization is an extreme expression of such improved receptibility and should be considered as an encouraging sign for conception. Fertil Steril 1992;57:139-42
The phenomenon of polypronuclear fertilization is accepted as a common, though undesirable, outcome of in vitro fertilization (IVF). Various authors reported its occurrence in IVF programs with an incidence of2% to 10%. 1- 3 The impression that fertilization and conception rates are exceptionally high in treatment cycles with such polypronuclear fertilization have stimulated us to investigate this phenomenon in our program.
MATERIALS AND METHODS Out of 832 consecutive IVF treatment cycles performed between April 1986 and March 1990, 646 successful oocyte retrievals were performed. Four different stimulation protocols were used:
Received December 21, 1990; revised and accepted September 4, 1991. * Reprint requests: Abraham Golan, M.D., Obstetrics and Gynecology, Assaf Harofeh Medical Center, Zerifin 70300, Israel. Vol. 57, No. 1, January 1992
1. Human merwpausealgonadotropin (hMG) (135 cycles): hMG (Pergonal; Teva, Petah Tikva, Israel) was started on day 3 at three ampules per day. 2. Early folicular D-Trp6 fhMG (213 cycles): An intramuscular injection of 3.2 mg of D-Trp6 luteinizing hormone-releasing hormone (LH-RH) (Decapeptyl; Ferring, Malmo, Sweden) was administered between days 1 and 3 of the menstrual cycle . When ovarian suppression was confirmed (estradiol [E 2 ] ~30 pgfmL and no follicle cyst on ultrasound) hMG treatment (3 ampules daily) was commenced (usually on day 18). 3. MidlutealD-Trp6 fhMG (227 cycles): The same protocol as in group 2 starting in the midluteal phase (usually day 22 to 24). 4. Ultrashort D-Trp6 fhMG (71 cycles): hMG was started as described in group 1. On day 2, a daily dose of 0.5 mg of D-Trp 6 LH-RH was administered intramuscularly for 3 consecutive days (days 2 to 4). In all our groups hMG was administered at three ampules per day and individually adjusted. Ovarian stimulation was monitored by daily E 2 , progesterone, Golan et al.
Polypronuclei indicate more IVF conceptions
139
and LH serum levels, and by ovarian ultrasonography. Human chorionic gonadotropin (10,000 IU, Chorigon; Teva) was given when the leading follicle was ~18 mm in diameter. Oocytes were retrieved vaginally under ultrasonographic control in the majority of the cases (95%), and the rest were retrieved laparoscopically. Up to four embryos were replaced and the rest cryopreserved. The suction pressure at oocyte retrieval was 120 to 160 mmHg, rarely increasing to 200 mmHg. The oocytes were inseminated 1 to 4 hours later using a semen concentration of 150,000 to 350,000 sperm/mL culture medium in center well plates. Fertilization was determined 18 to 22 hours later after mechanical removal of cumulus cells and the observation of two pronuclei. The X2 test with a continuity correction was used for statistical analysis.
RESULTS Out of the 646 cycles with oocytes retrievals, polypronuclear fertilization occurred in 40 cycles (in 36 patients). These cycles yielded 315 oocytes and 192 embryos out of which polypronuclear fertilization was found in 56. Among all the cycles with retrieved oocytes (646), the incidence of cycles with polypronuclear fertilization was 6.2%. The 56 polypronuclear embryos were 2.8% of the total of 1,987 embryos obtained. In the hMG group, the polypronuclear fertilization occurred in 45% of the cycles and was 2.1% of the total number of embryos. In the early follicular DTrp6/hMG group, the polypronuclear fertilization occurred in 3.8% of the cycles and was 1.3% of the total number of embryos. In the midluteal D-Trp6I hMG group, the polypronuclear fertilization occurred in 8.4% of the cycles and was 4% of the total number of embryos. In the ultrashort D-Trp6/hMG group, the polypronuclear fertilization occurred in 9.9% of the cycles and was 4.7% of the total number of embryos (Table 1). The rates of polypronuclear Table 1
fertilization in the various treatment protocol groups did not significantly differ (X 2 test, P = 0.1). In 12 cycles (one-fifth of the 40 polypronuclear fertilization cycles), there was more than one polypronuclear fertilization. Two triploid embryos were observed in 7 of the cycles, 4 triploid embryos in 2, a triploid and a tetraploid in 1, a triploid and a pentaploid in 1, and 2 hexaploids in 1 cycle (totaling 28 polypronuclear embryos in 12 cycles). The majority of the polypronuclear fertilizations occurred in mature oocytes. Of the 315 oocytes, 289 (92%) were mature and only 26 immature. Polypronuclear fertilization occurred in mature oocytes in 36 cycles and in immature oocytes in 6, (2 of the cycles comprised of both mature and immature oocytes). The average preinsemination oocyte incubation time in the polypronuclear group (2.34 hours) did not differ significantly from that of the normal fertilization group (2.66 hours) (P = 0.8). The vast majority of the patients with polypronuclear fertilization (33 of the 36 patients) suffered from unexplained infertility. Male factor was the cause of the infertility in two of the patients, and only one patient had a tubal cause for her infertility. In 36 of the 40 cycles, at least one embryo was replaced. The polypronuclear embryos were not replaced. Of a total of 221 patients aged ::::;;30 years, 17 had polypronuclear fertilizations. Twenty-eight cases of polypronuclear fertilization occurred among a total of 425 patients > 30 years of age. The difference was not statistically significant (P = 0. 7). Fertilization rate differed significantly between the polypronuclear fertilization cycles and the rest. Of the 315 oocytes, 192 fertilizations occurred (a fertilization rate of 61%) as compared with a fertilization rate of 36.6% in the normal cycles (of 4,442 oocytes 1,625 fertilizations) (P < 0.00001, X2 test) (Table 2). Pregnancy rate in the study group was also significantly higher. In the 40 polypronuclear fertiliza-
Incidence of Polypronuclear Fertilization Polypronuclear fertilization
Treatment protocol HMG Early follicular D-Trp6/hMG Midluteal D-Trp6/hMG Ultrashort D-Trp6 /hMG Total
140
Golan et al.
No. cycles
No. embryos
No. cycles
Percentage of cycles
No. embryos
Percentage of embryos
135 213 227
327 699 749 212 1,987
6 8 19 7 40
4.45 3.75 8.37 9.86 6.19
7 9 30 10 56
2.1 1.3 4.0 4.7 2.8
71
646
Polypronuclei indicate more IVF conceptions
Fertility and Sterility
Table 2
Fertilization and Pregnancy Rates Cycles with oocyte retrieval
No. of oocytes
No. of embryos
Fertilization rate
Clinical pregnancies
%
Polypronuclear fertilization Normal fertilization
Pregnancy rate/oocyte retrieval
Pregnancy rate/embryo transfer
%
%
40
315
192
60.9"
19
47.5b
sa·
604
4,442
1,625
36.6"
119
19.6b
28.8.
P < 0.00001. p < 0.0001. c p < 0.006. a
b
tion cycles, 19 clinical pregnancies occurred, with a pregnancy rate of 47.5% per cycle resulting in oocytes, compared with 119 pregnancies of the other 606 cycles, with a rate of 19.64% (P < 0.0001, X2 test) (Table 2). The pregnancy rate per embryo transfer in the polypronuclear group was 19 of 36 (53%) as compared with 119 of 444 (28.8%) in the normal fertilization cycles (P < 0.006). Three more patients of the polypronuclear group conceived in further IVF cycles. Among the 12 cycles with multiple polypronuclear fertilizations, 7 ended in pregnancy (58.5%) and an additional pregnancy occurred in a consecutive cycle. DISCUSSION
The most interesting finding in this study is the fact that fertilization and conception rates were significantly higher in cycles with polypronuclear fertilizations. This observation has been recently reported. 4•5 Increased fertilizing capacity of the sperm or improved oocyte receptibility could be the explanation. Polyspermy is believed to be the major factor involved in the occurrence ofpolypronuclear fertilization. Few explanations have been suggested for the increased incidence of polyspermy in vitro: (1) early insemination, (2) increased number of sperms surrounding the oocyte, and (3) delayed insemination. Sathananthan and Trounson6 observed a delayed release of cortical granules in polyspermic oocytes indicating delayed cortical maturation of the oocytes, eventually being the cause of polyspermy. In normal monospermic fertilization, cortical granules are released into the perivitaline space, decondense and swell, then interact with the inner layer of the zona pellucida and initiate a change in this area that prevents any further penetration by sperm. These authors suggested that polyspermy could be prevented by incubation for a few hours (up to 6 hours) Vol. 57, No. 1, January 1992
before insemination, thus improving cortical maturation.7 The large number of motile sperm surrounding the oocyte in IVF as compared with the smaller number in vivo has also been suggested as a possible etiological factor. Wolf et al. 8 clearly demonstrated that an increase in sperm concentration is directly related to an increase in the incidence of polyspermy. Plachot et al. 2 demonstrated a lower rate of polyspermy when sperm quality was reduced. The limit of 50,000 motile sperm per 1 mL of culture medium was suggested by Sathananthan and Trounson6 in an effort to reduce the incidence of polyspermia. Diamond et al., 3 however, did not find any effect of different sperm concentrations (125,000 to 500,000 per oocyte). Unlike previous reports by Sathananthan and Trounson6 and Trounson et al. 7 and Van der Ven et al., 9 Rudak et al. 1 found polypronuclear fertilization to predominantly occur in mature oocytes. They reported that a third of their polypronuclear fertilization occurred in postmature oocytes and none in immature oocytes. This seems to be in close agreement with our finding that polypronuclear fertilization occurs in mature rather than in immature oocytes. Plachot et al. 2 found that polyspermy is reduced when the preinsemination incubation period is short (
