Intracytoplasmic sperm injection in cases with a history of in vitro fertilization failure

Asian J Androl 2003 Mar; 5: 69-72             

Keywords: intracytoplasmic sperm injection; in vitro fertilization; male factor infertility; fertilization failure

Abstract

Aim: To evaluate the effect of intracytoplasmic sperm injection (ICSI) in the management of cases with a history of conventional in vitro fertilization (IVF) failure. Methods: Two groups of patients, 19 with normal semen parameters and a history of IVF failure (metaphase II oocytes: 0~30 %) and 28 with severe male factor infertility received ICSI technology during the same period. Ovarian stimulation was achieved by conventional procedure. Transvaginal ultrasound-guided oocyte collection was done 35~37 h after human chorionic gonadotrophin (hCG) injection. Only metaphase II oocytes were selected for microinjection. Results: Fertilization was achieved with ICSI in all the patients. The fertilization rate (75.6 %21.1 % vs. 73.9 %19.2 %), cleavage rate (85.1 %19.3 % vs. 82.7 %22.1 %), clinical pregnancy rate per embryo transfer cycle (31.6 % vs. 28.6 %) and implantation rate per embryo (15.3 % vs. 14.4 %) did not differ significantly between the two groups. Conclusion: ICSI is a valuable method for couples with a history of IVF failure. These patients may have a similar ICSI result as in severe male infertility.

1 Introduction

Intracytoplasmic sperm injection (ICSI) technique has become the standard treatment for infertility caused by male factors, as sperm parameters do not apparently affect the outcome of this technique and can be used successfully to treat couples who have failed conventional in vitro fertilization (IVF) or have too few spermatozoa for IVF [1]. Ziebe et al. [2] treated couples with non-male factor infertility having no or low fertilization in a previous IVF cycle. When they randomly subjected half of the retrieved oocytes to ICSI and the remaining half to conventional IVF, they obtained much better fertilization with ICSI and a pregnancy rate of 29 % per embryo transfer (ET). However, some centers has reported that ICSI was less successful in couples with unexplained failed IVF [3, 4] and the authors suggested that the poor outcome probably represented intrinsic oocyte defects that ICSI might not be able to overcome. We performed this study to evaluate the outcomes of ICSI in the treatment of patients with previous IVF failure and compare them with those undergoing ICSI for severe male infertility.

2 Materials and methods

Fourty-seven infertile couples were enrolled in our IVF program with written informed consent to receive ICSI technique. They were physically and mentally healthy with a normal karyotype. The study was approved by the Institutional Review Board. Patients were classified into 2 groups: Group 1 consisted of 19 couples with normal semen parameters and a history of fertilization failure in conventional IVF (MII oocytes: 0~30 %), the causes of infertility being anovulation (n=5), tuboperitoneal factors (n=5) and idiopathic problems (n=9); Group 2, 28 severe male factor infertile couples with very poor semen parameters, including 7 obstructive azoospermia. Semen analysis was done according to the World Health Organization manual [5].

In Groups 1 and 2, the ages of the female partners were similar (31.43.6 and 28.72.4 years, respectively; meanSD, P>0.05); so were the ages of the male partners (33.45.7 and 30.16.9 years, respectively, P> 0.05).

Patients were stimulated with a standard down-regulation and ovarian stimulation protocol and 7 patients had their stimulation regimens modified during their cycle. High responders and patients with polycystic ovary syndrome (n=3) were switched to dual suppression with oral contraceptives and Triptorelin (Pharma Biotech-Signes, France) intramuscularly. Low responders (n=4) had their dosage of Triptorelin halved. Follicular growth monitoring with ultrasonography and serum estradiol measurement was begun on stimulation day 3 and thereafter every 1~3 days as indicated. Oocytes were aspirated 35~37 h after human chorionic gonadotrophin (hCG, Profasi, Serono, Switzerland) administration by a transvaginal ultrasonographic procedure. Progesterone and/or hCG were used as the luteal support.

Following follicular aspiration and oocyte cumulus-corona complexes (OCCC) collection, the OCCC were cultured for 3~4 h in microdroplets of 10 % synthetic serum substitute (SSS, Irvine Scientific, USA) supplemented human tubal fluid (HTF, Irvine Scientific) medium under light mineral oil (embryo-tested, Medi-Cult, USA). The OCCC were then exposed to 60~80 IU of hyaluronidase (Medi-Cult) per milliliter in HTF- HEPES before the cumulus-corona complex was removed from the oocyte by repeated aspiration in and out with a hand-drawn Pasteur pipette. Upon completion, the oocytes were washed 3 times in HTF medium (containing 10 % SSS) and observed under an inverted microscope at 200 magnification. Each oocyte was assessed for the presence of the first polar body or absence of a germinal vesicle, as well as the integrity. Only mature MII oocytes were selected for micromanipulation. All cultures were maintained at 37 in a humidified atmosphere of 5 % CO₂.

On the day of aspiration, semen samples were assessed for volume and sperm concentration, motility and morphology. Motile spermatozoa were isolated from the ejaculates using either isolate gradient (Irvine Scientific) or swim-up method. Prepared sperm were layered over with 0.3~1.0 mL of sperm wash medium (HTF medium containing 10 % SSS). The tube was capped loosely and placed in an incubator (37 and 5 % CO₂) at 45o angle for 45~60 minutes. Then 0.5 mL of the upper-phase fluid was transferred into a tube for counting. In severe oligoasthenospermic ejaculates or samples from microepididymal sperm aspiration (MESA), they were washed and sedimented so that individual sperm could be selected for injection. When less than 50,000 spermatozoa were present in the samples, they were concentrated in 5 mL of HTF-HEPES medium and transferred directly to the dish.

The entire procedure was carried out on the heated stage of an inverted microscope equipped with Hoffman Modulation Optics (Leica DM IRB, Germany). The Narishige micromanipulators (IM-9B, IM-9C, Japan) systems were filled with mineral oil (M-8410, Sigma, USA). The microtools for ICSI (30 angle) were purchased from the In Vitro Microtool Inc., USA. Every procedure was performed by a fixed embryologist.

Before ICSI, 1uL of sperm suspension was diluted with 5 mL of 7 % polyvinylpyrrolidone (PVP; Irvine Scientific) solution in HTF-HEPES medium placed in the middle of a tissue culture plastic dish (Falcon 3002/1006, USA). Each oocyte was then placed in the plate surrounding the central droplet with each oocyte in its own 5 µL droplet of HTF-HEPES medium containing 5 % SSS. The droplets were then covered with mineral oil. A maximum of one or three oocytes were placed in the injection dish to avoid exposure to the HTF-HEPES medium for more than 10 minutes. The dishes were placed in the incubator with a lid until ready for use.

During ICSI, a single motile sperm with normal morphology was forcefully immobilized by touching the tail with the tip of the pipette 1~3 times. The oocyte was held by gentle suction with the first polar body oriented at 7 oclock. Before the sperm was pushed into the oocyte, the cytoplasm was aspirated repeatedly until a cytoplasm outflow was seen.

After ICSI, the injected oocytes were washed 3~5 times using equilibrated culture medium and incubated in drops of 50 mL in a tissue culture dish overlaid with light mineral oil. The dish was placed in a 5 % CO₂ atmosphere at 37 .

The injected oocytes were examined for the presence of pronuclei and polar bodies 12~17 h after ICSI. If two pronuclei were present, the oocyte was considered to have normal fertilization. Embryonic development was evaluated twice, at 24 h and at day 2 or 3. Embryos were graded as good [equal-size blastomeres with no or minor fragmentation (5 %~20 % blastomeres fragmented )] or poor [unequal-size blastomeres or major fragmentation (>20 % blastomeres fragmented)][3]. A maximum of two to three embryos (according to the age and the embryo quality) was transferred into each patient.

A b-hCG test was performed 14 days after ET. When two consecutive tests (at 2-day intervals) showed elevated b-hCG level, pregnancy could be considered. Pregnancy was confirmed when ultrasonograph 28 days after ET revealed a sac or fetal heart.

Data were expressed as meanSD. Statistical significance was determined with use of Student's t-test and chi square test as appropriate. P<0.05 was considered significant.

3 Results

All the oocyte retrieval cycles resulted in fertilization. The amount and days of FSH administration, the peak estradiol level and the endometrial thickness did not differ significantly between the two groups (Table 1).

Table 1. Comparison of basic conditions between the two groups. Group 1: with a history of previous IVF failure, Group 2: severe male factor infertility.

From the Table 2, it can be seen that the normal fertilization rate, the cleavage rate, the pregnancy rate per ET cycle and the implantation rate per embryo, etc. were similar between the two groups. In Group 1, there were 1 miscarriage and 1 ectopic pregnancy and in Group 2, 2 miscarriages and 1 ectopic pregnancy.

Table 2. Comparison of ICSI results between the two groups. Group 1: with a history of previous IVF failure, Group 2: severe male factor infertility. OCCC = oocyte cumulus-corona complexes.

In addition, when we compared the ICSI outcomes of the patients with complete fertilization failure (n=9) with those of the remaining patients (n=10), no differences in the fertilization rate (74.5 %22.2 % vs. 78.2 % 19.0 %; P>0.05), the cleavage rate (83.5 %11.5 % vs. 87.1 %12.2 %; P>0.05), the pregnancy rate (33.3 % vs. 30.0 %; P>0.05) and the implantation rate (15.8 % vs. 14.9 %; P>0.05) were found.

4 Discussion

Our results are in agreement with those of Svalander et al. [7], but differ from Miller et al. [4], who reported that patients with apparently normal semen parameters having a history of IVF failure had a significantly lower pregnancy rate than those without the history. The age difference in the women of the two groups might account for the disparity in the results. In the present series, 3 of the 9 infertile patients with no demonstrable cause had a successful pregnancy with ICSI, which was not significantly different from patients with known causes of infertility; it was similar to the results of Bukulmez et al. [8]. With oocyte donation in patients with previous IVF failure, Borini et al. [9] achieved a pregnancy rate similar to that seen in male factor infertility, suggesting that most patients with repeated fertilization failure probably have oocyte defects that can be bypassed only by donation.

The presence of chromosomal abnormalities may provide an explanation for the lower pregnancy rates of couples with a history of conventional IVF failure [10]. Most studies, however, have not described any changes in their subsequent ovarian stimulation protocols, whereas we did. It is conceivable that the differences may be attributed, at least in part, to an enhanced oocyte quality due to improvement in ovarian response. Theoretically, patients having a history of fertilization failure in conventional IVF may have a more serious oocyte or sperm defect than those having achieved fertilization, however, when the two groups were compared, we found similar outcomes of ICSI. Although the cycles were few in our study, the results may suggest that ICSI can overcome undetected oocyte or sperm defects. Moomjy et al. [11] evaluated the implications of complete fertilization failure after ICSI and concluded that it did not preclude successful fertilization and pregnancy in a later ICSI treatment.

In addition to occult gamete abnormality, the cause of fertilization failure in conventional IVF may include disturbances in sperm, oocyte, sperm-zona pallucida (ZP) penetration, sperm-oolemma fusion, etc. [12]. Obvious-ly, ICSI can improve the outcome of these types of infertility as the procedure bypassed the ZP and oolemma. The combination of semen analysis with specific sperm function tests may be worthwhile for the selection of patients for IVF or ICSI [13].

In conclusion, our study confirms that ICSI is an effective method for couples having failed conventional IVF. These patients can acquire the same fertilization rate, cleavage rate, clinical pregnancy rate and implantation rate as those with severe male infertility.

References

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[10] Munne S, Alikani M, Tomkin G, Grifo J, Cohen J. Embryo morphology, developmental rates, and maternal age are correlated with chromosome abnormalities. Fertil Steril 1995; 64:382-91.
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[13] Liu DY, Baker HWG. Evaluation and assessment of semen for IVF/ICSI. Asian J Androl 2002; 4: 291-5.

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Correspondence to: Prof. Zhi-Ling LI, Reproduction Center, First Affiliated Hospital, Medical College, Shantou University, Shantou 515041, China.
Tel: +86-754-893 2477
E-mail: lizhilin@pub.shantou.gd.cn
Received 2002-11-11   Accepted 2003-02-19