HUMAN STUDIES AND ISSUES

Collection and Preparation of Gametes

Oocytes

Human ICSI commonly involves collection of multiple in vivo matured oocytes after ovarian superovulation induced by pituitary gonadotropins. Stimulation protocols typically involve either gonadotropin-releasing hormone (GnRH) agonists or antagonists to suppress release of luteinizing hormone (LH), which can cause premature ovulation. Follicle-stimulating hormone (FSH) is used to stimulate multiple follicles, and human chorionic gonadotropin (hCG) may be used for final maturation of oocytes. The drug, dose and timing of gonadotropin administration will vary according to individual decisions of the clinician and the specifics of the patient’s case. For instance, some women are over suppressed by long duration administration of the GnRH agonists, and others may respond rapidly to initial stimulation. Some women respond better to LH suppression with antagonists of GnRH, rather than agonists. In addition, approximately 0.5% to 2% of superovulation treatments result in ovarian hyperstimulation syndrome, which may be very painful for the patient.²

Most commonly, multiple in vivo–matured oocytes are utilized. Collection is facilitated by either ultrasonographically guided transvaginal aspiration or laparoscopic retrieval. “Ultrasonically guided follicular aspiration is shown to be superior to laparoscopic oocyte recovery as far as ovarian accessibility and complication rate are concerned.”³

Spermatozoa

If spermatozoa cannot be harvested by ejaculation, then surgical extraction must be performed. Spermatozoa blocked by obstructive azoospermia can be removed by various microsurgical approaches, such as testicular sperm extraction (TESE). TESE is the most common technique to diagnose the cause of azoospermia and to obtain sufficient tissue for sperm extraction to be used either fresh or as a cryopreserved specimen. It involves one or multiple small biopsies of testis tissue and therefore is called testicular fine needle aspiration (TFNA). When a needle and syringe are used to puncture the skin to aspirate a sperm specimen, the procedure is called percutaneous epididymal sperm aspiration (PESA). PESA is popular because it can be performed repeatedly at low cost. PESA, like TFNA, can be completed without a surgical incision; however, it may not yield a suitable sample. Microsurgical epididymal sperm aspiration (MESA) involves direct retrieval of sperm from individual epididymal tubules and is performed under a microscope. It is completed by isolating the tubes and then aspirating the fluid. MESA limits damage to the epididymis while avoiding blood contamination of its fluid and yields high quantities of motile sperm that can be readily frozen and thawed for subsequent IVF treatments.

There no longer seem to be any categories of male factor infertility that cannot be treated with ICSI. Even for men with azoospermia caused either by obstruction or by germinal failure, ICSI may be performed successfully. The only failures will be in azoospermic men who have neither spermatozoa nor spermatids retrievable from the testis, but these men comprise a small percentage of the cases with severe male factor. The source of the spermatozoa and the cause of the sperm defect appear to have no effect on the success of the procedure, whether the spermatozoon is epididymal, fresh or frozen, testicular, ejaculated, or from the testicles of men with severe defects in spermatogenesis. Maturation arrest, Sertoli cell-only, cryptorchidism, chemotherapy and mumps do not appear to have a major impact on the pregnancy rate. Of all the factors studied in couples where the male is severely infertile or azoospermic, the only factor that seems to matter (as long as spermatozoa are retrieved) is the age of the wife and, to a considerably lesser extent, her ovarian reserve. Extensive genetic and paediatric follow-up studies of ICSI pregnancies have revealed no increased risk of congenital malformation (2.6%), no increased risk of de-novo autosomal abnormalities, and a 1.0% risk of sex chromosomal abnormalities.⁴

Fertilization

Fertilization can be achieved through a number of means, including “classical” IVF, gamete intrafallopian transfer (GIFT), intracytoplasmic sperm injection (ICSI), and various other methods of micro-manipulation.

Classical IVF relies on laboratory fertilization, and GIFT relies on normal physiologic fertilization in vivo. With both techniques, normal spermatozoa with the ability to swim, penetrate, decondense, and fertilize the oocyte are needed. Techniques of zona manipulation— zona drilling, zona dissection, and subzonal injection (SUZI)—all carry the risk of poorer embryonic development and/or the potential for multiple sperm fertilization of the one oocyte (polyspermy) but were developed to address the poor results with IVF and GIFT in cases of severe male factor infertility.⁵

ICSI was developed by Gianpiero Palermo in Belgium,⁶ resulting in the birth of the first child in 1992.⁷ Subsequently, a large amount of experimental and clinical data have been amassed. ICSI involves injection of a single spermatozoon into the cytoplasm of the oocyte. The oocyte is held in position by suction through a fine, rounded glass pipette. A single spermatozoon is immobilized by tail crushing or chemical retardation before aspiration into an extremely fine, sharp glass pipette and then insertion though the zona pellucida. Concerns were expressed initially about the potential to produce offspring from “inferior” sperm and the opportunity to transmit genetic abnormalities.

There is an ongoing discussion regarding conflicting data on malformation rate in children born after intracytoplasmic sperm injection (ICSI). A prospective, multicentric, control cohort study was done in Germany. Fifty-nine centres prospectively recruited pregnancies before the 16th week of gestation, which were included in the study if they were ongoing beyond this time. Children were examined according to a standardized procedure. A control cohort of children conceived spontaneously was taken from a prospective birth registry (Mainzer Modell), where children were examined according to the exact same criteria as the ICSI cohort. Major malformation rate was calculated, based on data of all liveborn and stillborn children, as well as on all spontaneous and induced abortions, beginning with the 16th week of gestation. In the ICSI cohort, 8.6% of infants (291/3372), and in the control cohort 6.9% of infants (2140/30940), had a major malformation. This resulted in a crude relative risk (RR) of 1.25 (95% confidence interval 1.11-1.40). There was no influence of sperm origin on major malformation rate in children born after ICSI. There is an increased risk for a child born after ICSI to have a major malformation compared with a child that has been spontaneously conceived. Based on knowledge of the early developmental steps following ICSI, as well as on data of conventional IVF in general, it is assumed that this increased risk is due to parental factors causing the infertility, which has led to ICSI in the first place.⁸

Other studies have not supported these findings and are discussed later in this chapter.

After micro-manipulation (i.e., ICSI and other techniques), the oocytes are cultured in vitro using defined medium and observed for early embryonic development. Developing embryos are graded, sorted, and transferred either fresh or frozen, or discarded.

The use of cytoplasmic transfer as an assisted reproductive technique has generated much attention and criticism. The technique involves the injection of cytoplasm from a healthy oocyte into a recipient oocyte considered unviable and includes the transfer of donor mitochondria. The consequences are the possible transmission of two mitochondrial (mt)DNA populations to the offspring. This pattern of inheritance is in contrast with the strictly maternal manner in which mtDNA is transmitted after natural fertilization and ICSI.⁹

Embryo Culture

With human ART, embryo culture and cryopreservation have resulted in an increased efficiency and flexibility. Commonly early-cleavage-stage embryos are transferred into the uterus despite the recognition that the embryo would not be expected in the uterine environment for at least 2 or 3 more days. This may result in a uterine environment that is not ideal for the embryo, perhaps associated with poor uterine clearance or an abnormal hormonal milieu. In addition, synchrony between the embryo and the uterus is poor. Oocytes fertilized and then cultured in vitro may be subjected to culture conditions that have affected the ability of the embryo to grow, and the technique also assumes that all oocytes, fertilized and in vitro–matured, have a similar ability to develop. Pregnancy rates are three times higher when in vivo–derived blastocysts (obtained by uterine flushes in fertile women) are transferred, compared with in vitro–derived early-cleavage-stage embryos (as with IVF for infertile women).¹⁰ Culturing embryos to the blastocyst stage in vitro has recently been associated with significant advantages. First, embryo morphology at the blastocyst stage is a predictor of pregnancy rates.¹⁰ Second, the most viable embryo within the group of cultured embryos may be selected.¹⁰ Third, the problems of multiple pregnancies may be addressed by examination of the probability that a single embryo will be transferred successfully before the transfer is performed. It also has been hypothesized that the uterine environment may not be as hostile to a blastocyst as to an earlier-cleavage-stage embryo.¹⁰

Furthermore, as the score of the blastocysts obtained using sequential media is directly related to implantation and pregnancy rates, it is possible to determine which patients should be offered a single blastocyst transfer, thereby addressing the issue of twins conceived through ART.¹¹

Transfer

Embryos commonly are transferred at the 4- to 8-cell stage (day 2 or 3 after fertilization), although a current trend is for embryo culture and transfer at the blastocyst stage ¹¹ because this approach could be expected to improve implantation rates. In addition, embryo grade is correlated with pregnancy rates.¹²

Embryos are mostly transferred through the vagina in a non–embryo-toxic catheter, through the cervix and into the uterus. The number of embryos transferred depends on embryo quality and age of the woman. More embryos are transferred into older women, with less success.¹³^–¹⁵

Preimplantation genetic diagnosis (PGD) to identify chromosomally normal embryos has recently become a development applied to embryos before transfer. This technique involves the biopsy of a blastomere from an 8- to 16-cell embryo. Gender, genetic disorders, and chromosomal number and structure are commonly evaluated.

Despite its novelty, preimplantation genetic diagnosis has become an alternative to traditional prenatal diagnosis, allowing the establishment of only unaffected pregnancies and avoiding the risk of pregnancy termination. In addition, preimplantation genetic diagnosis is presently applied for much wider indications than prenatal diagnosis, including common diseases with genetic predisposition and preimplantation human leukocyte antigen typing, with the purpose of establishing potential donor progeny for stem cell treatment of siblings. Many hundreds of apparently healthy, unaffected children have been born after preimplantation genetic diagnosis, presenting evidence of its accuracy, reliability and safety. Preimplantation genetic diagnosis appears to be of special value for avoiding age-related aneuploidies in patients of advanced reproductive age, improving reproductive outcome, particularly obvious from their reproductive history, and is presently an extremely attractive option for carriers of balanced translocations to have unaffected children of their own.¹⁶

On occasion, zona-assisted hatching may be employed to help the embryo break out of the zona pellucida. Previous implantation failure in otherwise normal patients may suggest this problem. Chemicals, lasers, and mechanical manipulation all have been used to create a breach in the zona.¹⁷

Assisted hatching entails the opening or thinning of the zona pellucida before embryo transfer in order to improve the results of in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). The technique can be performed mechanically, chemically or with a laser beam. A piezoelectric method has also been described. Meta-analyses of randomised trials have shown that assisted hatching increases the clinical pregnancy, implantation and on-going pregnancy rates in patients with poor prognosis for IVF and ICSI, particularly those with repeated implantation failure. The technique is not without risks, and has been associated with an increased incidence of monozygotic twinning. Nevertheless, it remains an invaluable tool in assisted reproductive technology.¹⁸

This may be even more relevant in cases of in vitro maturation of oocytes.

Immature oocyte recovery followed by in-vitro oocyte maturation and in-vitro fertilization is a promising new technology for the teatment of human infertility. The technology is attractive to potential oocyte donors and infertile couples because of its reduced treatment intervention. Immature oocytes were recovered by ultrasound-guided transvaginal follicular aspiration. Oocytes were matured in vitro for 36-48 h followed by intracytoplasmic sperm injection (ICSI). Embryos were cultured in vitro for 3 or 5 days before replacement. Assisted hatching was performed on a day 5 blastocyst stage embryo. Embryo and uterine synchrony were potentially enhanced by luteinization of the dominant follicle at the time of immature oocyte recovery. Mature oocyte and embryo production from immature oocyte recovery were similar to the previous IVF results of the patients. A blastocyst stage embryo, produced as a result of in-vitro maturation, ICSI, in-vitro culture and assisted hatching, resulted in the birth of a healthy baby girl at 39 weeks of gestation.¹⁹

Pregnancy

Approximately 30% of IVF pregnancies produce twins, 5% triplets, and less than 1% more than three children. Multiple pregnancies carry high risks for the woman and the fetuses.

Multiple gestation pregnancy rates are high in assisted reproductive treatment cycles because of the perceived need to stimulate excess follicles and transfer excess embryos in order to achieve reasonable pregnancy rates. Perinatal mortality rates are, however, 4-fold higher for twins and 6-fold higher for triplets than for singletons. Since the goal of infertility therapy is a healthy child, and multiple gestation puts that goal at risk, multiple pregnancy must be regarded as a serious complication of assisted reproductive treatment cycles. The 1999 ESHRE Capri Workshop addressed the psychological, medical, social and financial implications of multiple pregnancy and discussed how it might be prevented. Multiple gestations are high risk pregnancies which may be complicated by prematurity, low birthweight, pre-eclampsia, anaemia, postpartum haemorrhage, intrauterine growth restriction, neonatal morbidity and high neonatal and infant mortality. Multiple gestation children may suffer long-term consequences of perinatal complications, including cerebral palsy and learning disabilities. Even when the babies are healthy they must share their parents’ attention and may experience slow language development and behavioural problems. Current data indicate that the average hospital cost per multiple gestation delivery is greater than the average cost of in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) cycles. Prevention is the most important means of decreasing multiple gestation rates. Multiple gestation rates in ovulation induction and superovulation cycles can be reduced by using lower dosage gonadotrophin regimens. If there are more than three mature follicles, the cycle should be converted to an IVF cycle, or it should be cancelled and intercourse should be avoided. In IVF cycles two embryos can be transferred without reducing birth rates in most circumstances. Embryo reduction involves extremely difficult decisions for infertile couples and should be used only as a last resort. Assisted reproductive treatment centres and registries should express cycle results as the proportion of singleton live births; twin and triplet rates should be reported separately as complications of the procedures. Reducing the multiple gestation pregnancy rate should be a high priority for assisted reproductive treatment programmes, despite the pressure from some patients to transfer more embryos in order to improve success. If nothing is done, public concern may lead to legislation in many countries, a step that would be unnecessary if assisted reproductive treatment programmes and registries took suitable steps to reduce multiple pregnancy rates.²⁰

There are data regarding the possible influences of extended embryo culture to the blastocyst stage as well as zona pellucida manipulation on the incidence of monozygotic multiples. This is interesting, as one aim of extended culture with embryo selection is to minimize the multiple pregnancy rate. We report, to our knowledge, on the first case of monozygotic twins and monozygotic triplets after ICSI and the transfer of two blastocysts. Monozygotic multiples after ICSI and blastocyst transfer and the resulting problems are another reason to encourage the transfer of only one blastocyst. In our opinion, the incidence of 5.9-8.9% monozygotic multiple occurrence after ICSI and blastocyst transfer reported in the literature requires that patients are informed of the uncertainties until this phenomenon and its risk factors are better understood.²¹

Current Status of Human Intracytoplasmic Sperm Injection

Since the publication of the first papers on the use of ICSI for oligozoospermia in 1992 and 1993, much scientific work has been applied to extending its application to virtually every type of male infertility.^4,^7,^22,²³ In 1995 Nagy²⁴ confirmed that the most severe cases of oligoasthenoteratozoospermia produced the same pregnancy rates as for mild cases of male factor infertility, which were no different from those for men with normal spermatozoa undergoing conventional IVF. Then it was demonstrated that the way in which the spermatozoa are pretreated before ICSI is immaterial, and that any method for aspirating the spermatozoa into an injection pipette and transferring them into the oocytes is adequate.²⁵ It also was reported that fertilization failure was always related either to poor egg quality or to sperm nonviability.²⁶ It appeared that no matter how severe morphologic defect, or with the most severe motility defect or the smallest number of spermatozoa in the ejaculate (pseudoazoospermia), no negative effect on the pregnancy rate was observed with ICSI. Only absolute immotility of ejaculated or epididymal spermatozoa lowered the fertilization rate, and this was found to be not due to the immotility of the spermatozoon but rather to its nonviability. Completely nonmotile spermatozoa that were viable were still capable of normal fertilization and pregnancy rates.²⁷

ICSI then took another leap forward with the development of sperm aspiration and extraction techniques, which allowed couples in which the male was absolutely azoospermic to achieve pregnancy rates no different from those in which the male had a normal sperm count.²⁸ The first successful attempts at sperm aspiration combined with ICSI were reported in 1994.²⁹ Conventional IVF with aspirated epididymal spermatozoa yielded a pregnancy rate of only 9% and a delivery rate of only 4.5%, whereas ICSI with aspirated epididymal spermatozoa in men with congenital absence of the vas deferens (CAVD) yielded a pregnancy rate of 47% and a delivery rate of 33%. Furthermore, no difference in pregnancy rates was found with use of epididymal spermatozoa retrieved for any cause of obstruction, whether it was failed vasoepididymostomy, CAVD, or simply irreparable obstruction.³⁰

However, this breakthrough for men with CAVD brought with it a serious problem. It was soon discovered that CAVD is caused by mutations on the cystic fibrosis transmembrane conductance regular gene (CFTR) located on chromosome 7. Although now this is taken for granted, in 1992 it was a startling discovery…. This discovery meant that all patients and their wives undergoing sperm aspiration with ICSI for CAVD required careful genetic screening for cystic fibrosis, and if the wife was a carrier (4% risk of carrier status in the general population), then the embryos should undergo preimplantation genetic diagnosis using polymerase chain reaction, so that only healthy embryos would be replaced.²⁷

The first case of successful preimplantation embryo biopsy for cystic fibrosis, on the embryo of a man who had undergone MESA and ICSI for CAVD, was reported in 1994.³¹“The use of MESA and ICSI for CAVD led to intense molecular study of the genetic mystery of how the condition of CAVD is transmitted via defects in the cystic fibrosis gene.”²⁷

Soon after the MESA-ICSI procedure was developed in 1994, it was discovered that testicular spermatozoa could fertilize as efficiently as ejaculated spermatozoa and also result in normal pregnancies.

In this study (May 1 until August 31, 1994) a total of 15 azoospermic patients suffering from testicular failure were treated with a combination of testicular sperm extraction (TESE) and intracytoplasmic sperm injection (ICSI). Spermatozoa were available for ICSI in 13 of the patients. Out of 182 metaphase II injected oocytes, two-pronuclear fertilization was observed in 87 (47.80%); 57 embryos (65.51%) were obtained for either transfer or cryopreservation. Three ongoing pregnancies out of 12 replacements (25%) were established, including one singleton, one twin and one triplet gestation. The ongoing implantation rate was 18% (six fetal hearts out of 32 embryos replaced).³²

TESE truly revolutionized the treatment of infertile couples with azoospermia. The development of TESE meant that even patients with zero motility of the epididymal spermatozoa or of ejaculated spermatozoa, or even men with no epididymis, could still have their own genetic child, so long as there was normal spermatogenesis. It also meant that surgeons could easily perform a testicle biopsy, enabling the retrieved spermatozoa to be used for ICSI without the need for the microsurgical expertise required to perform a conventional MESA procedure.²⁷

It also was demonstrated that epididymal spermatozoa, despite fairly weak motility, could be frozen and, after thawing, yield pregnancy rates no different from those obtained with freshly retrieved epididymal spermatozoa.

In seven patients who did not become pregnant following microsurgical epididymal sperm aspiration (MESA) and intracytoplasmic sperm injection (ICSI), a subsequent ICSI was performed using previously cryopreserved super-numerary epididymal spermatozoa without re-operating on the husband. During the original MESA procedure a mean sperm concentration of 12.3 × 10(6)/ml was achieved. The supernumerary spermatozoa were cryopreserved for later use. After thawing frozen epididymal spermatozoa a mean concentration of 1.9 × 10(6) spermatozoa/ml was obtained in straws containing a total volume of sperm suspension of 250 microliters. From 68 intact oocytes injected with frozen-thawed epididymal spermatozoa, a two pronuclear fertilization rate of 45% and a cleavage rate of 82% were obtained. A total of 17 embryos were replaced in the seven patients, resulting in two ongoing singleton pregnancies and one twin delivery. Six embryos were cryopreserved. In conclusion, it would appear mandatory to cryopreserve supernumerary spermatozoa during a MESA in order to avoid subsequent further scrotal surgery.³³

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