Delayed fertilization during in vitro fertilization and embryo transfer cycles: analysis of causes and impact on overall results

Transcription

1 FERTILITY AND STERILITY Copyright" 1989 The American Fertility Society Printed on ocid free paper in U.S.A. Delayed fertilization during in vitro fertilization and embryo transfer cycles: analysis of causes and impact on overall results Sergio Oehninger, M.D. Anibal A. Acosta, M.D. Lucinda L. Veeck, M.L.T. (A.S.C.P.) Simonetta Simonetti, M.S. Suheil J. Muasher, M.D. The Howard and Georgeanna Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Medical College of Hampton Roads, Norfolk, Virginia This study evaluated possible causes of delayed fertilization during in vitro fertilization (IVF) cycles, its repetitiveness, and its influence on IVF results in 23 patients (27 cycles) with delayed fertilization of ~1 preovulatory oocyte(s). In 15 cycles, reinsemination with husband's semen was performed at 18 hours. Possible causes of delayed fertilization were oocyte defects (10 cycles, 37.0%), sperm defects (4 cycles, 14.8%), oocyte and sperm defects (4 cycles, 14.8%), and no detectable gamete defects (9 cycles, 33.3%). Overall fertilization rate was 47.9%. No pregnancies were observed in 10 patients with one embryo transferred. Recurrence rate of delayed fertilization per patient was 17.3%; overall ongoing pregnancy rate/cycle was 10.3%. Although repetitiveness of delayed fertilization is low, it seems to impact negatively on IVF results. Fertil Steril52:991, 1989 The success of fertilization in in vitro fertilization (IVF) relies upon several factors, including gamete(s) adequate in quality and quantity, strict laboratory control, and timed oocyte insemination. In the Norfolk IVF program the overall fertilization rate for preovulatory oocytes, including male factor patients, has been maintained at approximately 85%.1 We have reported that the incidence of failed fertilization represents approximately 5% of attempts and that sperm abnormalities can be identified as causal factors in a high percentage of cases. 1 Absence of distinct pronuclear formation by 18 to 20 hours after oocyte insemination, followed by the late appearance of pronuclei and extrusion of the second polar body, has been referred to as delayed fertilization. 2,3 Delayed fertilization can occur spontaneously or after oocyte reinsemination, Received May 18, 1989; revised and accepted July 26, Reprint requests: Sergio Oehninger, M.D., Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, 825 Fairfax Avenue, Norfolk, Virginia usually with a new, fresh semen sample. Trounson and Webb 2 reported that reinsemination did not enhance fertilization rates compared with controls, in whom spontaneous, delayed fertilization had occurred without reinsemination. On the other hand, Ben-Rafael et a1. 3 observed a fertilization rate of approximately 27% after reinsemination, associated with a high polyspermic rate (29%). They achieved pregnancy in 7/66 patients when embryos derived from reinseminated oocytes were transferred along with those fertilized on initial insemination. These authors therefore recommended reinsemination to increase the overall fertilization rate. Boldt et a1. 4 also reported that reinsemination was associated with substantially higher fertilization rates (40% to 70%), thereby increasing the number of embryos available for transfer. The potential value of oocyte reinsemination was reflected in the birth of one infant subsequent to the transfer of embryos derived solely from successful reinsemination. The objectives of this study were (1) to investigate the possible gamete (oocyte and/or spermato- Oehninger et ai. Delayed fertilization during IVF 991

2 zoa) defect(s) associated with delayed fertilization to try to establish a causative role; (2) to evaluate the results in cases where reinsemination was performed versus cycles in which "spontaneous" delayed fertilization occurred, and (3) to determine the repetitiveness of this phenomenon and its overall influence on IVF results. MATERIALS AND METHODS During Norfolk series 25 to 33 (October 1986 to December 1988) 867 patients underwent 1462 IVF cycles (1,321 transfers). Twenty-three patients (2.6% of patients) were identified as having had one or more cycles with delayed fertilization for a total of 27 cycles (1.8% of cycles) and an overall total of 58 cycles (including all other cycles without delayed fertilization). Delayed fertilization was defined as the absence of distinct pronuclei 18 to 20 hours after oocyte insemination, with delayed appearance of male and female pronuclei spontaneously or after oocyte reinsemination, as discussed below. Only preovulatory oocytes (metaphase I and II at aspiration) were considered. Patients in the study group were treated by IVF for primary infertility (n = 15) or secondary infertility (n = 8). Their mean age was 35.6 ± 0.8 (x ± standard error [SE]) years, with a range of 30 to 40. The primary indications for IVF were tubal factor (8 patients), tubal and male factor (6 patients), endometriosis and male factor (2 patients), dysovulatory and male factor (2 patients), male factor (3 patients), and endometriosis (2 patients). In 13 patients a male factor was involved, alone or in combination with female factors, as the cause of infertility. All semen parameters except sperm morphology were evaluated according to the established World Health Organization (WHO) criteria.5 Morphology was evaluated by the strict criteria of the Norfolk andrology laboratory.6 Immunologic investigations (of semen and serum antisperm antibodies) and the hamster ova/human sperm penetration assay (SPA) were performed according to published protocols. 7,8 Diagnosis of a male factor was established when sperm concentration was <20 X 106/mL, progressive motility < 50% (WHO criteria),5 normal morphology < 14%,6 and/or penetration rate in the SPA < 10%,8 The specific semen and systemic abnormalities found in these men were severe teratozoospermia and abnormal SPA (five patients), severe teratozoospermia with asthenozoospermia and abnormal SPA (one patient), oligoasthenozoospermia with severe teratozoospermia and abnormal SPA (two patients), mild teratozoospermia (four patients), and systemic (serum only) antisperm antibodies (one patient). Severe teratozoospermia was defined as <4 % normal forms ("poor" prognosis pattern), whereas mild teratozoospermia was defined as 4 % to 14% normal forms ("good" prognosis pattern),6 The female partners (normally menstruating women) were assessed by the basic infertility workup required in the Norfolk program1 and also by determinations of serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) on cycle day 3, We have reported that these values are helpful in predicting stimulation response and IVF outcome (normal laboratory range for FSH and LH: 5 to 20 miu/ml).9,1o Seven patients had abnormal endocrine profiles in addition to other primary causes of infertility. These abnormalities were elevated FSH levels (>20 miu/ml) in four patients and LH/FSH > 2/1 in three other patients. The IVF procedures used for ovarian stimulation (a combination of 2 FSH/2 human menopausal gonadotropin [hmg]/human chorionic gonadotropin [hcg] or "pure" FSH/hCG), sperm preparation, oocyte classification, insemination and culture, and embryo transfer in the Norfolk program have been described. ll,12 In our experience all of these stimulation protocols have demonstrated provision of preovulatory oocytes with identical fertilization rates.13 Each patient was further categorized according to her plasma estradiol (E2) response pattern. ll Oocyte retrieval was performed by laparoscopy or by the transvaginal ultrasonically guided approach, as reported. 14 In an effort to improve stimulation response and/or IVF results, we switched patients who returned to the program to different stimulation protocols (modification of the basic combination gonadotropin protocol, pure FSH in low or high doses, 2 to 6 ampules/d15,16) or to concomitant use of a gonadotropin-releasing hormone agonisty Other corrective measures implemented in subsequent attempts included modification in the sperm insemination concentrations, where indicated,13 and the use of a cryopreserved donor semen sample (one patient). For patients with normal sperm parameters, timed oocyte insemination was performed with the standard concentration of 50,000 motile sperm/ 992 Oehninger et al. Delayed fertilization during IVF Fertility and Sterility

3 Table 1. Stimulation Protocols and Serum E2 Response Patterns ill 27 Cycles with Delayed Fertilization Stimulation protocol 2 FSH/2hMG 6FSH 4FSH 2FSH GnRHa/2 FSH/2 hmg Serum E2 response pattern High Intermediate Low No. of cycles ml/oocytep After April 1987 the insemination concentration of male factor patients, especially those with teratozoospermia, was increased to 5~0,000 to 1,000,000 motile sperm/ml/oocyte, with a remarkable improvement in the fertilization rate.13 The oocytes were evaluated for signs offertilization at 12 to 20 hours after insemination (15 to 26 hours after retrieval). If they were judged to be unfertilized by 20 hours, (a) either they were transferred to a new culture dish containing fresh medium and were reinseminated, usually with a freshly obtained semen sample; ifthis was not feasible, the initially washed motile sperm fraction was used if it still displayed good motility. (These reinseminated oocytes were reexamined 12 to 18 hours later for evidence of fertilization.) Reinsemination was performed in 9 ofthe 13 male factor patients and in 4 patients without semen abnormalities. Or (b) if other mature oocytes from the cohort had fertilized, or if the quality and quantity of the sperm population attached to the zona pellucida or in close contact with the oocyte investments was judged to be adequate, they were observed for 10 to 12 more hours to assess whether spontaneous delayed fertilization could occur. Results of the study group were compared with those of the overall population attempting IVF during the same time frame. Statistical analyses were performed by x2 and the student's t-test where appropriate. P levels < 0.05 were considered significant. Results are expressed as the mean ±SE. RESULTS The stimulation protocols used and the serum E2 response patterns for the 27 cycles with delayed fertilization are shown in Table 1. In 19 cycles the E2 response was intermediate, in 2 cycles it was high (associated with a multi-follicular ovarian response and a high basal serum LH/FSH ratio) 15,18 and in 6 cycles it was low (in 4 cases with a ~erimenopausal pattern of high basal serum FSH levels).19 In the 27 cycles, a total of 123 preovulatory 00- cytes (61 metaphase I, 62 metaphase II) were recovered. Thirty-nine of the 123 oocytes (31.7%) showed delayed fertilization. In 15 of these cycles reinsemination with husband's semen had bee~ performed, whereas in 12 cycles spontaneous-delayed fertilization was observed. Ofthe oocytes displaying delayed fertilization, 61.5% were metaphase I and 38.4 % were metaphase II at aspiration. The overall fertilization rate per oocyte for the 27 cycles was 47.9% (59/123 oocytes), significantly lower than that of controls (5027/5818, 86.4%; P < ). The polyspermia rate was 7.3% (9/123) versus 9.5% (477/5027) in controls, not significantly different. Gamete abnormalities found in these cycles were as follows. (Table 2): oocyte defects (10 cycles, 37.0%), either purely morphological (refractile bodies,20 vacuolar ooplasm) or associated with an abnormal endocrine profile (perimenopausal pattern,19 multi-follicular ovarian response associated with high serum E2 levels in the periovulatory period);15,18 sperm defects (4 cycles, 14.8%), severe impairment of sperm motility and/or morphology6 «4% normal sperm forms), combined with poor penetration rates in the SPA; a combination of oocyte and sperm abnormalities (4 cycles, 14.8%), and no gamete defects (unexplained; 9 cycles, 33.3%). The fertilization rates for each subgroup were significantly different (2 X 4 contingency table, P < 0.005), being lowest for the group with sperm defects (14.2%) and highest in the group with no ga- T~ble 2 Gamete Abnormalities Found in 27 Cycles with Delayed Fertilization No. of cycles Fertilization rate a % % Sperm abnormalities 4 (14.8) 4/28 (14.2) Oocyte defects 10 (37.0) 15/28 (53.5) Oocyte + sperm defects 4 (14.8) 13/32 (40.6) No abnormalities (unexplained) 9 (33.3) 27/35 (77.1)." The fertilization rate was significantly lower in the group with sperm abnormalities than in the other three groups. Oehninger et al. Delayed fertilization during IVF 993

4 Table 3 Analysis of Etiologic Factors in the Seven Patients Who Achieved a Pregnancy in the 58 Attempted IVF Cycles Patient Gamete abnormalities Sporadic/recurrent Female diagnosis Male diagnosis 1 None (unexplained) Recurrent (2/2 cycles) Tubal Normal Normal 2 None (unexplained) Sporadic (1/2 cycles) Tubal Sporadic (1/3 cycles) Endometriosis Normal 3 None (unexplained) 4 None (unexplained) Recurrent (2/5 cycles) LH/FSH> 2 Normal FSH> 20 mid /ml Normal 5 Oocyte Sporadic (1/4 cycles) 6 Oocyte 1 cycle LH/FSH> 2 Systemic immunologic 7" Sperm 1 cycle Normal Severe oligospermia " Pregnant in second cycle using donor semen. mete or endocrine abnormalities (unexplained, 77.1 %). Individual x 2 analyses showed that the fertilization rate from the group with sperm abnormalities was significantly lower than from the groups with oocyte defects (P < 0.005), oocyte and sperm defects (P < 0.03), and unexplained (P < ). The mean number of embryos transferred in the 27 cycles was 2.7 ± 0.1, and the mean number of blastomeres at transfer was 3.0 ± 0.4, values not significantly different from those of controls. Only three pregnancies were achieved, for an ongoing pregnancy rate per transfer of 11.1 %. No pregnancies were observed in 10 patients (13 cycles) in whom only one embryo was transferred (derived from a mature oocyte that displayed delayed fertilization). All three pregnancies were achieved after the transfer of embryos derived both from oocytes initially fertilized and those displaying delayed fertilization. The pregnancy rate with only one embryo transferred in the control group was 12%. Besides these 27 cycles, patients from the study group had 31 additional cycles without evidence of delayed fertilization, for an overall total of 58 cycles. The overall ongoing pregnancy rate/transfer was 10.3% (6 pregnancies in 58 cycles). (Another patient achieved a pregnancy using donor semen in a case of severe male factor infertility). This figure was lower than in the control group (17.6%; 233 ongoing pregnancies/1321 transfers), although not significantly different. Table 3 presents an analysis of the etiologic factors encountered in the seven patients who became pregnant in the 58 attempted IVFcycles. The recurrence rate of delayed fertilization was 17.3%/patient (4/23) in the 58 cycles (8 cycles with delayed fertilization in 11 attempted cycles). In three of these patients no gamete abnormalities were detected; in the remaining patient a repetitive oocyte problem was observed, associated with fer- tilization delay in combination with a 50% polyspermic rate. Reinsemination versus Spontaneous Delayed Fertilization Of the 15 cycles in which the oocytes (n = 23) were reinseminated, the overall fertilization rate was 42.3% (33/78). The success rate of reins emination was 33.8% (23/68 oocytes that had not fertilized initially). Gamete abnormalities were equally distributed among this group, although the absence of any detectable gamete defects was most common (33.3% of cycles) (Table 4). In the 12 cycles with spontaneous delayed fertilization (n = 26 oocytes), the overall fertilization rate was 57.7% (26/45). The success of observation or watchful expectancy3 was 45.7% (16/35 oocytes that had not fertilized initially). Oocyte defects were found in 58.3% of these cycles. DISCUSSION A few studies have investigated the value of reinseminating oocytes that failed to fertilize within 15 to 20 hours after initial insemination or the option of adopting a watchful expectancy that delayed spontaneous fertilization would occur. 2-4 This is the first investigation of the possible causes of this phenomenon, its prognostic significance, and its repetitiveness. Oocyte defects were observed in 37.0% of the cycles with delayed fertilization; this could be the postulated cause for its occurrence. Veeck 20,21 established that oocytes with atypical morphology fail to fertilize in a high percentage of cases. She reported that oocytes with refractile bodies (multivesicular structures with lipid droplets, associated with dense granules, small vesicles, and fibrillar material) fertilized in only 2% of the cases, com- 994 Oehninger et al. Delayed fertilization during IVF Fertility and Sterility

5 Table 4 Comparison of Oocyte Reinsemination versus "Spontaneous" Delayed Fertilization Oocytes Oocytes fertilized/ Overall fertilized/ reinseminated or No. of No. of fertilization "initially" "observed" Major gamete ongoing cycles rate inseminated oocytes abnormalities pregnancies % % % % Oocyte reinsemination Unexplained 1 (33/78) (10/78) (23/68) (33.3) "Spontaneous" Oocyte defects 2 delayed fertilization (26/45) (10/45) (16/35) (58.3) All 3 pregnancies were achieved after transfer of embryos derived both from "initially" and "delayed" fertilized oocytes. pared with an 86% fertilization rate in controls (metaphase II oocytes with normal morphology). In addition, preovulatory oocytes with atypical characteristics of the first polar body at collection demonstrated lower fertilization rates (50% to 75%) together with an increase in abnormal fertilization (13% to 19%) compared with controls.21 It is tempting to speculate that there is a wide spectrum of oocyte atypias or dysfunctions that result in delayed fertilization, polyspermic fertilization, or complete failure of fertilization. The oocyte itself appears to control, at least partially, crucial stages of the fertilization process such as establishing the block to polyspermia, promoting sperm nuclear head decondensation, and resuming an active metabolic state during activation.22 Oocytes of poor morphological or functional quality may fail totally or partially during this process, leading to fertilization abnormalities. Whether these oocytes of poor quality are intrinsically abnormal or are the result of exogenous ovarian stimulation and an unfavorable endocrine milieu remains to be determined.15,18,19 Plachot et al. 23 reported that the rates of chromosomal anomalies and mosaicism are drastically increased in delayed fertilization; maternal age but not the type of infertility or the type of stimulation was associated with the chromosome anomalies that they found. Spontaneous fertilization or fertilization after reinsemination always raises the possibility of oocyte immaturity3 and inadequate maturation diagnosis at retrieval, leading to improperly timed oocyte insemination. In our embryology laboratory we routinely use Veeck's cumulus spreading technique for correct identification of the oocyte nuclear state.21 Knowledge of maturational status (prophase I, metaphase I and II) allows for a more accurate timing of insemination, and Veeck has reported a slight increase in normal fertilization rates along with a slight decrease in triploidy with this technique.21 Because the block to polyspermia is a reversible phenomenon,24 it could also be postulated that some oocytes could undergo premature release of cortical granules content with an initial block to polyspermia, followed by a reversal of this phenomenon, thus allowing for delayed fertilization to occur. This hypothetical mechanism might occur in aging oocytes and/or oocytes with other dysfunctions. Severe sperm abnormalities, alone or in combination with oocyte defects, were associated with delayed fertilization in 29.6% of the cycles. This may suggest an involvement of the male gamete in some cases of delayed fertilization. This is not surprising, because we have diagnosed sperm defects in 70% of patients with failed fertilization in IVF.l A low-motility and a high percentage of abnormal sperm forms have been associated with poor fertilization rates in IVF.13,25 Also poor and failed penetration in the SPA have been observed in patients with failed IVF. 8 On the other hand, the presence of serum or semen antisperm antibodies has not been shown to affect IVF results negatively in our program.25 The involvement ofthe male gamete in the fertilization process is apparently very complex and other subtle sperm dysfunctions could lead to abnormal or delayed fertilization. Sperm binding to the zona pellucida is a critical initial step of fertilization, and we have reported that patients with failed fertilization show significantly decreased tight binding to the zona than patients with successful fertilization in IVF, as evaluated by the hemi-zona assay.25 Whether dysfunctional sperm/ zona pellucida interactions, failure, or retardation of the sperm triggering of oocyte activation,22 or abnormal interactions between oocyte cytosolic factors and a spermatozoon could be one of the under- Oehninger et ai. Delayed fertilization during IVF 995

6 lying causes of delayed fertilization remains to be established. In 33.3% of the cycles with delayed fertilization, no gamete defects were observed and no major endocrine abnormalities could be diagnosed (unexplained). It is hypothesized that subtle gamete dysfunction(s) at an unknown stage of the fertilization process may have contributed to the delay in these patients. In 17.3% of the patients (72.8% of their cycles) delayed fertilization occurred as a repetitive phenomenon. This finding suggests that in some patients intrinsic and permanent gamete defects are the subjacent cause. In the remaining cases, delayed fertilization was sporadic, raising the possibility that intercurrent factors played a predominant role (changes in gamete microenvironment? endocrine milieu? in vitro culture conditions?). The latter possibility appears to be extremely unlikely because of the strict quality control and rigorous culture conditions that rule our IVF laboratory. Similar success rates were observed in reinsemination and spontaneous delayed fertilization (33.8% and 45.7%, respectively). However, one should not be misled by this high -efficiency of reinsemination since, by study design, all these patients achieved fertilization. On the contrary, the real success rate of reinsemination for the control group was only 2.8% (23 oocytes with second insemination success per 814 total oocytes with initial failure of fertilization). This figure is more in agreement with that reported by Trounson and Webb,2 who found a 13% fertilization rate following reinsemination using a freshly collected semen sample. That the overall fertilization rates were low in the groups with reinsemination and delayed fertilization (42.3% and 57.7%, respectively) points to the fact that delayed fertilization may be an epiphenomenon of a more profound fertilization defect. Even though three pregnancies were achieved in the 27 cycles with delayed fertilization, in all cases there was a mixture of transferred embryos derived both from oocytes initially fertilized and from 00- cytes with delayed fertilization. That no pregnancies were observed when only one embryo, derived from an oocyte with delayed fertilization, was transferred, plus the low-ongoing pregnancy rate/ transfer of all IVF cycles in this population, suggests that the reproductive performance of these patients is impaired. In conclusion: cycles with delayed fertilization are associated with demonstrable gamete defects in 66% of the cases. Although the repetitiveness of the phenomenon is low, it seems to have a negative impact on IVF results. Acknowledgments. The authors thank Richard Scott, M.D., for helpful discussions during the preparation of the manuscript, Charlotte Schrader, Ph.D., for editorial assistance, and Ms. Debbie Jones for computer assistance. REFERENCES 1. 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7 Oehninger S, Scott R, Muasher SJ, Acosta AA, Jones HW, Jr, Rosenwaks Z: Effects ofthe severity oftubo-ovarian disease and previous tubal surgery on the results of in vitro fertilization and embryo transfer. Fertil Steril51:126, 1989 Muasher SJ: Stimulation protocols for patients with "atypical response." Ann NY Acad Sci 541:82,1988 Hofmann GE, Toner JP, Muasher SJ, Jones D, Liu H-C, Jones GS: High-dosage follicle stimulating hormone (FSH) ovarian stimulation in low responder patients with high basal serum FSH for in vitro fertilization. (Abstr. 052) Presented at the 44th Annual Meeting of the American Fertility Society, Atlanta, Georgia, October 10 to 13, Published by the American Fertility Society, Birmingham, Alabama, 1989, p S18 Droesch K, Muasher SJ, Brzyski RG, Jones GS, Simonetti S, Liu H-C, Rosenwaks Z: Value of suppression with a gonadotropin-releasing hormone agonist prior to gonadotropin stimulation for in vitro fertilization. Fertil Steril51:292, 1989 Romeu A, Muasher S, Acosta AA, Liu H-C, Rosenwaks Z: Hormonal and follicular behavior of patients displaying multifollicular ovarian response (MOR) when undergoing IVF with different stimulation protocols. (Abstr. PP-5) Presented at the 5th World Congress on In Vitro Fertiliza- tion and Embryo Transfer, Norfolk, Virginia, April 5 to 10, Published by the American Fertility Society in the Program Supplement, 1987, p Jones GS, Muasher SJ, Rosenwaks Z, Acosta AA, Liu H-C: The premenopausal patient in in vitro fertilization: the use of gonadotropin-releasing hormone. Fertil Steril 46:885, Veeck LL: Atlas of the Human Oocyte and Early Conceptus. Baltimore: Williams Wilkins, 1986, p Veeck LL: Oocyte assessment and biological performance. Ann NY Acad Sci 541:259, Yanagimachi R: Mammalian fertilization. In The Physiology of Reproduction, Edited by E Knobil, JD Neill. Raven Press, 1988, p Plachot M, de Grouchy J, Junca AM, Mandelbaum J, Salat-Baroux J, Cohen J: Chromosome analysis of human 00- cytes and embryos: does delayed fertilization increase chromosome inbalance? Hum Reprod 3:125, Gulyas BJ: Electron microscopic observation on advanced stages of spontaneous polyspermy in rabbit zygotes. Anat Rec 179:285, Acosta AA, Oehninger S, Morshedi M, Swanson RJ, Scott R, Irianni F: Assisted reproduction in the diagnosis and treatment of the male factor. Obstet Gynecol Surv 44:1, 1989 Oehninger et al. Delayed fertilization during IVF 997