Abstract
Up to the 1980s the ability to treat couples with male subfertility was limited. Treatable conditions included gonadotrophin therapy for men with hypogonadotrophic hypogonadism (which is estimated to be present in 1 in 200 infertile men), and the treatment of autoimmunity with high dose steroids with its side effects and complications (i.e. avascular necrosis of the head of the femur) [1].
10.1 Introduction
Up to the 1980s the ability to treat couples with male subfertility was limited. Treatable conditions included gonadotrophin therapy for men with hypogonadotrophic hypogonadism (which is estimated to be present in 1 in 200 infertile men), and the treatment of autoimmunity with high dose steroids with its side effects and complications (i.e. avascular necrosis of the head of the femur) [1]. Obstructive azoospermia could sometimes be treated by epididymal or vasal micro-surgery with moderate results for caudal blockages, but poor results were obtained for proximal blockages, and even if spermatozoa appeared in the ejaculate, pregnancies were rare. Disorders of sexual function could sometimes be treated, and toxic causes (maybe 1 in 500 men) removed [2].
However, about 70% of men who had abnormalities of sperm numbers or quality had no diagnosable cause, and were treated in an uncontrolled way, without evidence of efficacy of any of the following methods [3].
Androgens were used either as rebound therapy where spermatogenesis suppression was hoped to be followed by “rebound” improvement, after the hormone is stopped, or as low-dose testosterone administration, aiming to improve epididymal maturation of sperm [4].
Attempts to improve sperm quantity by increasing gonadotropin secretion by administering clomiphene citrate [5] or injections of gonadotropins [6] were given to “stimulate” spermatogenesis. In men where the presence of infection/inflammation was suspected (raised leucocyte count in the semen), antibiotics and anti-inflammatory drugs were administered. Vitamins and antioxidants, herbs, amino acids, and minerals such as zinc and selenium were also administered empirically, based on a theoretical beneficial effect. Cold testicular douches were also used. Many treatments were recommended on the basis of a report of some quantitative improvement of semen factors, but of course this could always be due to spontaneous improvement, or the phenomenon of “regression towards the mean.” Pregnancy rates were not used as an endpoint, so no treatment was evidence based. Artificial insemination with partner’s sperm and insemination with donor was widely used.
When the possibility of using IVF in the management of male infertility emerged, there was no longer the need to make a specific diagnosis, nor the need to attempt to improve sperm quality, since only small numbers of spermatozoa were required to fertilize oocytes in vitro. It was a casual conversation over sandwiches in the Reproduction Medicine Clinic at Prince Henry’s Hospital (a multidisciplinary clinic established in 1977 attended by andrologists, gynaecologiosts, a urologist, and a counsellor), when David de Kretser suggested that maybe males with subfertility and decreased numbers of motile normal sperm could achieve fertilization in vitro, where they could not achieve this in vivo [7]. This led to Monash IVF establishing a “Male Factor Group,” which David chaired. He took a sabbatical for two months, and selected couples who may have benefited from his large infertility practice. Chris Yates (PhD student and embryologist) became the male factor scientist, Jillian McDonald the nurse co-ordinator, and I was the IVF clinician. Prior to this project, one of the prerequisites for IVF treatment (which at that time was mainly confined to the treatment of tubal disease) was to have a normal semen analysis. Alan Trounson had already attempted to apply IVF to unexplained subfertility [8] and male factor was yet another possible application for IVF, where men with low sperm counts, poor sperm motility, and those with an increased percentage of abnormally shaped sperm could all attempt to achieve a pregnancy. The technique used for preparing the sperm for IVF in the Melbourne program at the time is summarised by Alex Lopata: [9]
Husband’s semen was diluted in modified Tyrode’s solution and centrifuged. The sperm pellet was diluted again and centrifuged to obtain washed sperm pellet that was free of seminal plasma. A final sperm pellet dilution provided about 600,000 motile sperm in one ml of Tyrode’s, supplemented with albumin, was used for inseminating the oocyte.
The aim of male factor IVF was to separate the most motile sperm for incubation with the oocytes. Initially the “swim-up” technique was used, followed by a centrifugation–migration technique, with the sperm pellet being re-suspended and motile sperm isolated from uppermost portion of suspension. Later, by adding Ficoll (Pharmacia, Sweden), a density gradient interface was achieved, allowing only motile spermatozoa to pass across it. Albumin columns, glass wool columns, and Percoll gradients were all utilized as sperm selection techniques, aiming to obtain the best sample to add to the oocytes [10].
The use of microdrops for insemination was adopted with low volumes of semen of reasonable quality. It provided good results in mild to moderate male factor subfertility [11,12].
The availability of this technical development led Graeme Southwick (plastic and reconstructive surgeon/microsurgeon) and Peter Temple-Smith (research scientist) to surgically obtain such samples from seminiferous tubules using an operating microscope to recover small numbers of spermatozoa from men with obstructive azoospermia, which could then be used for inseminating oocytes in vitro obtained from their partners. The first baby born from sperm microsurgically recovered from a man with obstructive azoospermia after previous vasectomy, then utilizing IVF, was “Baby Joseph” [13].
10.2 Micromanipulation of Oocytes and Sperm
Even with the microdrop technique, many spermatozoa (tens of thousands) were still required. In order to allow smaller numbers of sperm to achieve fertilization, micromanipulation procedures were introduced (Figure 10.1). The two initial assisted fertilization procedures attempted were zona drilling and partial zona dissection (PZD). Unfortunately, although occasional fertilization and pregnancies were reported, neither of these methods was very successful [14,15]. Zona drilling involved making a hole in the zona pellucida of an oocyte, which was then incubated in a sperm suspension. Although this worked well in mice, it did not have much success in humans.
PZD, whereby a mechanical slit was made in the zona pellucida before they were incubated in a sperm suspension, was also attempted. Although fertilization was obtained by this method, monospermic and polyspermic fertilizations were as common as normal fertilization. Although some pregnancies and births occurred, inconsistent results meant that PZD was not widely applied clinically. Around the same time a few case reports were published on the next assisted fertilization procedure: subzonal insemination (SUZI), a micromanipulation technique involving the insertion of a few spermatozoa into the perivitelline space between the zona pellucida and the membrane of the oocyte.
Schematic representation of assisted fertilization procedures: PZD, SUZI, and intracytoplasmic sperm injection (ICSI), is shown in Figure 10.1.
The possibility of using sperm microinjection techniques was suggested as early as 1984 when Alan Trounson wrote, “The possibility of microsurgical fertilization procedures are also being investigated in our laboratory” [16].
The first “Microinjection trial” was commenced at the Infertility Medical Centre (later renamed Monash IVF) in 1987, co-ordinated by Ross Hyne (Figure 10.2).
Trounson and colleagues reported success in mouse oocytes with SUZI [17] and the technique was replicated in humans [18]. Unfortunately, the Minister for Health in Victoria in March 1988 requested that further use of SUZI be suspended (Figure 10.3). With the ban on SUZI in Australia, S. C. Ng who had visited Monash University produced the world’s first human birth using SUZI in Singapore in 1988 [19] and several other babies following in Australia and the UK [20–22].
10.3 Two Major Breakthroughs: ICSI and Testicular Sperm Aspiration
ICSI was developed in Brussels at the Centre for Reproductive Medicine of Vrije Universiteit Brussel (VUB), under the supervision of Andre van Steirteghem. He had introduced SUZI into the clinic for patients that had failed several cycles of conventional IVF. He obtained ethical approval from the VUB Hospital Ethical Committee under the condition that all pregnancies and children born would be thoroughly followed up, including prenatal diagnosis by either chorionic villous sampling or amniocentesis. Clinical SUZI was started at VUB in 1990 and a number of pregnancies and births occurred. This was from sub-zonal insemination of a few spermatozoa, which had been treated prior to enhance the acrosome reaction [23]. The technical procedure of SUZI is delicate and occasionally one of the sperm entered into the cytoplasm of the oocyte. However, in these cases of “failed SUZI,” they observed normal fertilization as well as embryo development. He called this procedure ICSI (Figure 10.4). In April 1991, a patient became pregnant after replacing a single ICSI embryo and she delivered on January 14, 1992 [23]. After the initial ICSI observations they continued SUZI and also included ICSI on some oocytes in most cycles. It rapidly became very obvious that the results in terms of fertilization were much more consistent after ICSI than after SUZI, and they obtained ethical approval for ICSI under the same strict protocol, with respect to following up of any pregnancies. After July 1992, the only mechanically-assisted fertilization procedure practiced at VUB was ICSI [24,25]. From late 1992, several ICSI workshops were held at VUB, and they taught many embryologists from around the world, which helped a lot with the dissemination of ICSI worldwide. Andre states that “the VUB’s openness to the world in showing ICSI was similar to the approach taken by the Melbourne groups for the introduction of conventional IVF.”
