Effect of age and cycle responsiveness in patients undergoing intracytoplasmic sperm injection

Vol. 68, No.1, Jul y 1997

FERTILITY AND STERILITY""

Printed on acid -free paper in U. S. A

Copyright c 1997 American Society for Reproductive Medicine Publis hed by Elsevier Science Inc.

Effect of age and cycle responsiveness in patients undergoing intracytoplasmic sperm injection Samer Alrayye s, M.D.*t Hasan Fakih, M.D.* Iqbal Khan, Ph .D.:!: Michigan State University, College of Medicine, and Sag inaw General Hospital-North, Saginaw, Michigan

Objective: To evaluate the effects of age and number of embryos available for transfer on pregnancy rate (PR) in couples undergoing intracytoplasmic sperm injection (lCSI ). Design: Retrospective study of patients undergoing ICSI for male factor infertility. Setting: Tertiary car e academic center. Patient(s): One hundred twelve patients < 37 years of age and 57 patients 2:37 years of age who underwent ICSI with uterine ET or tubal ET. Intervention: Compare cycles in which three embryos or fewer were available for transfer with those with four or more available embryos. Main Outcome Measure(s): Implantation, embryo availability, and pregnancy and miscarriage rates. Result(s): Younger patients did significantly better with regard to PR (47% versus 26%), implantation rate (11.4% vers us 6.6%), and ongoing PR (40% ver sus 19%). Patients in whom more than four embryos were transferred also did better than patients in whom three or fewer embryos wer e available for transfer, with an implantation rate of 14.2% versus 4.2%. In women < 37 years of age, 85% of cycles produced more than three embryo s, versus 61% in women 2:37 years of age. When cycles yielding three embryos or fewer were excluded, the younger group tended to do better, with an ongoing PR of 48% versus 33% for the older group, but the differences were not statistically sign ifican t. Both groups had similar number of embryos tra nsferred per cycle. Conclusion(s): Age affects cycle responsiveness, and the number of embryos available for transfer affects fertility in patients undergoing ICSI. Older women with good ovarian response, producing more than three embryos suitable for transfer , have a PR similar to younger patients. Cycles yielding three embryos or fewer have a poor prognosis, (Fertil Steril ® 1997;68:123-7. © 1997 by American Society for Reproductive Medicine.) Key Words: ICSI, age, number of embryos available for transfer

In 1978, when Steptoe and Edward (1) reported the first IVF pregnancy, IVF-ET was heralded as the biggest breakthrough for management of female infertility. Thousands of infants born through the Received Sept ember 9, 1996; revised and accepted Mar ch 6, 1997. * Division of Reproduct ive End ocrinology, Department of Obstetrics and Gynecology, Michigan State Univers ity , College of Medicine . t Repri nt requests: Samer Alrayyes, M.D., Division of Reproductive Endocrinology, 5400 Mackinaw, Suite 4100, Saginaw, Michigan 48604 (FAX: 517-792-3377). :j: IVF/Andrology Laboratory and First-IVF , Saginaw General Hospit al-North .

0015-0282/97/$17.00 PH S0015-0282(97)00071-X

application of this and other assisted reproductive technologies (ARTs) are a testimony to that achievement. Assisted reproductive technologies, however, have failed to alleviate male factor infertility. Fertilization rates with moderate sperm dysfunction re mained low whereas severe male factor infertility remained out of bounds of any therapeutic procedure. A glimpse of hope came with the introduction of microfertilization for t he treatment of male infertility, namely, partial zona dissection and subzonal insemination (SUZI) (2, 3). The fertilization rates by these procedures, however, remained < 20%. In 1992, Palermo et al. (4) reported the first successful pregnancy and birth after the use of intracy-

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toplasmic sperm injection (lCS!) in a couple with severe male factor infertility. The most beneficial effect of this procedure is the high fertilization rate achieved with even severely defective sperm characteristics. Research has been conducted to find factors that affect the success of ICSI. Sperm quality was found to be of little consequence, and most of the factors involved are related primarily to the female partner (5,6). Age has been shown to reduce fertility (7 -9), and a substantial number of infertility patients present in their late 30s or beyond. In this study, we investigated the effect of age on pregnancy rate (PR) and cycle responsiveness. We also studied cycles in which similar number of embryos were available for transfer in two different age groups and compared the effect of age. MATERIALS AND METHODS

One hundred twelve women <37 years of age, with severe male factor infertility, underwent 136 transfer cycles of ICSI performed because of poor sperm parameters (group A). Similarly, 57 patients, ~37 years of age, underwent 72 cycles of ICSI because of severe oligoasthenospermia (group B). Couples were selected for ICSI if they suffered from at least one of the following disorders: [1] very low fertilization « 15% in 10 oocytes) in a previous IVF cycle; [2] concentration of motile sperm <5 million in total ejaculate; [3] total absence of sperm; or [4] <4% normal morphology. All patients received GnRH agonist in the midluteal phase followed by 1M administration of hMG (Pergonal; Serono Laboratories, Inc., Randolph, MA), starting on the 3rd day of the menstrual cycle (10). When three or more follicles showed a mean diameter ~ 18 mm and serum E z exceeded 800 pg/mL (2,800 pmol/L), 10,000 IU hCG was administered. Thirty-six hours later, follicular aspiration was performed using ultrasound (US)-guided transvaginal aspiration. Both groups had similar luteal-phase support with oral P, 600 mg daily starting on the day of retrieval, and 50 mg 1M every other day starting after the day of transfer, increased to 50 mg 1M daily 1 week later and maintained for 6 weeks. One week after retrieval, 5,000 IV hCG 1M was administered. After aspiration, oocytes were identified immediately, rinsed free from blood, scored, and transferred into microdrops of Ham's F-I0 (Flow Laboratories, Irvine, CA) supplemented with Plasmatein (Alpha

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Alrayyes et aI. lCS1, age, and cycle responsiveness

Therapeutics, Los Angeles, CA). Mature oocytes were stripped of their cumulus-corona complex by using 80 Iu/mL hyaluronidase (Type VIII H3757; Sigma, St. Louis, MO) prepared fresh in human tubal factor-HEPES solution. Oocytes that showed expulsion of 1st polar body were used for ICSI (11). The injection and holding pipettes were prepared by pulling glass capillaries on the puller P-87 (Drummond; Sutter Industries, Novato, CA). With regard to the sperm injection pipette, the pulled capillary first was cut on a microforge (MF-9; Narashige Instrument, Tokyo, Japan) at an outer diameter of 7 to 8 j),m and an inner diameter of 4 to 5 us», A bevel of 48 to 50° was made on a microgrinder (EG-4; Narashige). A sharp spike was produced by a "gentle touch, touch, and pull" method on the microforge filament. By positioning the heater filament close to the shaft of the pipette (5 to 8 mm from the tip), an angle of 45° was produced. The micromanipulation system, NT88, was purchased from Narashige, USA. For sperm injection and oocyte holding, 1M-6 microinjectors were used. They were fitted with 800-/1L glass microsyringes. The glass syringe was filled with light mineral oil (M8410; Sigma). Similarly, the Teflon tubing, which connects the syringe to the glass micropipette holder (HI-4), was filled with oil. Care was taken to remove all air bubbles from the system before ICSI was performed. For a precise and smooth sperm injection, the Teflon tubing had to be kept as short as possible. Both the injection and the holding pipettes had to be in the same plane when sperm injection was performed. Both the stereomicroscope and the inverted microscope were fitted with heated stages (Cryogenic Concepts, Allentown, PA). A morphologically normal-looking motile sperm was immobilized by pressing the midpiece or tail region with the tip of the injection pipette. This immobile sperm was drawn tailfirst into the pipette. Oocytes then were injected (4). Embryo transfer was performed 48 to 72 hours after retrieval. Tubal embryo transfer (TET) was performed when at least one normal tube was encountered. There was one cancellation for a cycle that yielded two eggs that failed to fertilize in a 41-year-old patient (cancellation rate of 1.4% in that group). Institutional Review Board approval was obtained. We define clinical pregnancy as US documentation of gestational sac or ectopic pregnancy. We define ongoing pregnancy as a pregnancy> 20 weeks of gestation. Student's two-tail t-test was used for comparison of means and X Z for proportions.

Fertility and Sterility»

Table 1 Clinical Cha racteristics and Outcome of the Two Age Grou ps

P Grou p A Group B « 37 years) (2:37 years) value 31.3 :t 3.2 Averag e age (y)*t 112 No. of patien ts 136 No. of cycles Fertili zation rate (%) 70.1 Impl a nt aton rate (%)* 11.4 706 (5.2) No. of ETs*t 64 (47) No. of pregnancies*t 54 (40) No. of ongoing pregnanciest z Misca rriage rate (%)§ 15.6

39.7 :t 2.6 57 72 64.4 6.6 294 (4.1) 19 (26) 14 (19 .4) 26

0.001 0.2 0.02 0.01 0.004 0.004 0.28

* Values

are averages :t SD; P < 0.05. t Values in parentheses are percentages of ETs per cycle. t Values in parentheses are percentages of pregnancies per cycle. § Values in parentheses are percentages.

RESULTS

of cycles in women ;:0:: 37 years . Data are shown in Table 2. Effect of Age in Cycles Yielding More Than Three Embryos Suitable for Transfer

Both of the group s of women < 37 years and th e older group received similar number of embryos per cycle (6.0 and 5.5, re spectively, P = 0.115). The fertilization rate of the younger group was 72.0%, and the rate was 66.6% in women "2:37 years (P = 0.5). Tubal embryo transfer was performed in 68% of cycles of the younger age group ver sus 51% in women ;:0:: 37 years of age (P = 0.01). Pregnancy rate (58% ver sus 43%), ongoing PR (48% versus 33%), implantation rate (13.2% ver sus 10.2%), and miscarriage rate (15% versus 28%) were not significant at the 95% confidence interval. The multiple gestation rate of the younger group (31%) was similar to that of the older group (28%). Data are shown in Table 3.

Effect of Age

The younger age group had a higher number of embryos available for transfer , received an average of 5.2 ETs per cycle (P = 0.01), and had a higher PR than the older group (47% ver sus 25%, P = 0.004). Also higher for the group of women < 37 years of age was the implantation rate : 11.4% vers us 6.6% for women ;:0:: 37 years . The fertilization rate of the women < 37 years was 70.4%; it was 64.6% in the older group (P = 0.2). In th e younger age group, 62.5% of transfers were TET , versus 46.4% in women ;:0::37 years (P = 0.005). Data are shown in Table 1. Effect of Cycle Responsiveness

Cycles that yielded three embryos or fewer suitable for transfer were compared with those yielding four or more embryos . Women producing more than three embryos were younger than t hose producing three or fewer (P = 0.001), and their fertilization rate was significantly higher (71.8% versus 54.1%, P = 0.001). An average of 5.6 eggs were harve sted from cycles that yielded three or fewer embryos, versu s 15.1 eggs from the other group (P = 0.001). In those cycles producing more than three embryos, 65.7% of transfers were TETs , and in tho se cycles producing three or fewer , 51% of transfers were TETs (P = 0.02). Pregnancy rate, ongoing PR, and implantation rate were all significantly higher in th e group whose cycle yielded four or more embryos . The miscarriage rates were comparable between the two groups. Fifteen percent of cycles in women < 37 years produced three embryos or fewer, versus 39%

Vol. 68, No.1 , July 1997

DISCUSSION

Various authors (6) have shown that sperm parameters seem to play a small role with regard to progno sis. Pregnan cies have been achieved from sperm obtained from the testi s and epididymis (5), from occasionally motile sperm (12), and from t eratozoospermia (13). With the advent of ICSI , female factors seem to dictate prognosis. The decline of fertility with aging has been well documented. Approximately one third of women who defer pregnancy until their late 30s and approximately one half of tho se > 40 years of age will have an infertility problem. Tucker et al. (5) and Oehninger et al. (6) have found age to have a significant negative impact on embryo implantation, and sue-

Table 2 Effect of th e Embryo Availability on Pregnan cy Outcome

P > 3 ETs Average age* No. of cycles Fertilizati on rate (%)* Impl an t ation rate (% )* No. of ET s*t No. of pr egnan cies*:j: No. of ongoing pr egn an cies'tt Miscarriage rat e (%) §

33.3 :t 4.4 148 71.5 12.4 867 (5.9) 79 (53) 66 (45)

17

,;;3 ETs

36.0

:!:

5.8

value 0.01

60

53.3 3.3 133 (2.2) 4 (6.7) 2 (3.3) 50

0.001 0.04 0.01 0.002 0.002 0.089

* Values

are averages :t SD; P < 0.05. t Values in parentheses are percen t ages of ETs per cycle.

~ Values in parentheses a re per centages of pregnan cies per cycle. § Values in parentheses are percentages.

Alrayyes et al. ICSI, age, and cycle responsiveness

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Table 3 Effect of Age When More Than 3 Em bryos Are Availa ble for Tr an sfer Group A « 37 years) Average age (y)" No. of pati ents No. of cycles Fertiliz ati on rate (%) Impl an tati on rate (%)* No. ofETs*t No. of pr egnan cies*:t: No. of ongoing pregnancies*:t: No. of miscarriages (% )*§ Multiple gestation*1I

31.4 Z 3.1 95 106 73.6 13.2 634 (6.0) 61 (58) 52 (48) 9 (15) 19 (31)

Group B P years ) value

(~ 3 7

38.9 :!: 2.4 35 42 66.1 10.2 233 (5.5) 18 (43) 14 (33) 5 (28) 5 (28)

0.001 0.5 0.22 0.115 0.106 0.083 0.45 0.79

* Values are averages z

SD; P < 0.05. t Valu es in parentheses are per cent ages of ETs per cycle.

:j: Valu es in parentheses are percentages of pregnancies per cycle. § Values in parentheses are miscarriage rates. II Valu es in parentheses are multiple gestation rates.

cess of the ICSI procedure is dependent primarily on female factors. In our experience, we also have found this to be the case . On further review of our data, we identified cycles that yielded three or fewer embryos suitable for transfer and compared those with cycles yielding four or more. The results were very significant . Cycles yielding fewer than four embryos came from an older patient (36 years) and had a very low number of eggs harvested per cycle (5.6), a low fertilization rate (54.1%), and a PR of6.7%. It can be argued that the older ovary is less responsive and that its eggs may have a cytoskeletal defect that impairs fertilization. Thirty-nine percent of cycles (22/57) in women > 36 years fell into this category, as compared with only 15% in the younger age group (17/112). After excluding those cycles and comparing th e two initial age groups, the differences in the ongoing PR and fertilization rate became insignificant, even though the difference in age was significant (P = 0.001). Both received similar number of embryos per cycle (6 for the group < 37 and 5.5 for the group ~3 7 years), yielding 31% and 28% mul tiple gestation rate s, respectively (P = 0.79). All of the multiple births were twins except for one tri plet gestation. Balmaceda et al. (14), in a st udy of embryo implantation in oocyt e donation, achie ved an ongoing PR of 45%. The average age of recipients was 39 years and that of donors was 29. These researchers used high doses of P (100 mg 1M) and 6 to 8 mg of Ez daily. Meldrum (15), in a review of published data on ovarian and uterine aging in recipients of donated oocyte s, also had similar results. Meldrum (15) also found that women >40 years

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Alrayyes et al. l eSl, age, and cycle responsivene ss

need ed higher doses of P (100 mg 1M) to achieve a PR similar to that of younger patients and that if that dose was dropped to 50 mg, the PR dropped. From the above two st udies, it appears that the older uterus needs more P compared with a younger uterus to regain it s receptivity. We have used a high dose of P for both of our group s to negate th e negative effect of age on the uterus and enable us to study th e effect of age on embryo availability independent from uterine receptivity. Significantly more patients in the younger age group had TETs , and it can be argued that the higher PRs observed in this group are because of, at least in part, the method of transfer . However, when we analyzed the groups with more than three embryos available for transfer, both groups did as well, even though patients <37 years of age had significantly more TETs than the older group (P = o.on We conclude that female age has a detrimental effect on success of rCSI. That effect is directed mainly at ovarian responsiveness. Ovaries with a poor respon se produce poor-quality eggs. Fewer eggs are produced by older ovaries with a lower fertilization rate compared with eggs produced by younger patients. Consequently, fewer embryos suitable for transfer are produced that do not implant as well. According to our st udy, an older woman whose ovaries have less than six follicles seems to have a poor prognosis, because these follicles will yield a maximum of five eggs. With a fert ilization rate of approxima tely 55%, sh e can expect to have two to three embryos suitable for t ransfer. On the other hand, older women with good ovarian re sponse achieve a fertilization rate and a PR comparable to those of young er patients. Refining the stimulation protocols to increase ovarian responsiveness, assisted hatching, and reinsemination of those eggs that fail to fertilize may play a role in increasing the number of embryos available for transfer and improving the PRo REFERENCES 1. Ste ptoe PC, Edward RG. Birth after reimplantation of a hu -

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Fertility and Sterility"

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