The Journal of Reproductive Medicine for the Obstetrician and Gynecologist
Volume 11, Number 5, November 1973

CURRENT STATUS OF IN VITRO FERTILIZATION AND EMBRYO TRANSPLANTATION

EDWARD T. TYLER, M.D., RAFAEL TEJADA, M.D. and JAROSLAV MARIK, M.D.
The Tyler Clinic
Los Angeles, California 90024

Recently in a segment of the television series, "The Bold Ones", the plot involved transplantation of an embryo from one sister to another. Presumably the older married sister suffered rheumatic, valvular, cardiac pathology that precluded the possibility of her being able to successfully carry a pregnancy to term except at great risk. In making a case for embryo transplant, it was implied that adoption would be intolerable for this pregnant woman and that emotionally she would not be able to stand a "therapeutic abortion". In fact, as the plot unfolded, she would not even understand the term "therapeutic abortion". This situation led the doctor to tell the husband and wife that while successful embryo transplantations in humans have not as yet been reported, it is quite likely that Rock and others have already accomplished them. Grasping at this straw. the couple decided on the procedure. It then became necessary to find a suitable recipient. After an unsuccessful search, the woman s younger unmarried sister was finally suggested as a possibility and seemed a likely candidate in view of the fact that she seemed extroverted, emotionally stable. and what might be termed "disgustingly healthy". After initial astonishment and negative reaction to the idea. the sister finally agreed to the plan. With theoretical. assumed, adequate preparation of the recipient, surgery on both sisters was performed, with the ``blastocyst" removed from one uterus and "delicately" implanted in the other.

The plot of this program assumed a much more advanced state of the art than we have reached, and many of the formidable problems were simply glossed over or not even mentioned. The program did have merit in that it raised some of the emotional, ethical, medical, and legal problems of gamete and embryo manipulation in humans. In this story, much emphasis was placed on the emotional changes that took place in both donor and recipient and some legal problems were introduced (i.e. who does the baby really belong to?). But the story line was simplified by- having the younger sister have a spontaneous abortion. It was intimated that the sister's carelessness, possibly induced by emotional conflicts over the pregnancy, led to its termination.

Those of us involved in fertility problems recognize that scientifically there are many difficult problems involved in successful embryo transplants in humans. We also recognize that enough groundwork has already been laid to suggest that full-term development and delivery of embryos implanted into a human uterus after in vivo or in vitro fertilization elsewhere will be accomplished possibly within the next few years. This speculation must take into account the many scientific factors still unresolved, as well as the existing controversy concerning whether in vitro fertilization has yet actual been accomplished in humans.

Advances have taken place in animal husbandry-. For example, Chang, one of the pioneers in fertility research. reported a series of experiments with rabbits in 1951 [12] in which he demonstrated that successful transplantation could occur with a particularly good rate of success when the age in days of the ovum agreed with the interval in days after ovulation on the part of the recipient. Subsequently, Averill [l] confirmed these general results with ewes in 1957. In 1963, Nishikawa and associates [28] reported that, with suitable preparation of the uterine endometrium, normal pregnancies were obtained with transplanted ova in goats. These authors concluded that "daily injection of 10 mg of progesterone with a view to synchronizing the age of ova in days, suppressed estrus as long as the injection was continued. In most cases, estrus occurred on the second day after the last dosage. The duration of estrus was 1.5 to three days in such cases. One each of four ova (at the two- and eight-cell stages) recovered from two donors was transplanted. 10 to 30 minutes after recovery, into four recipients whose sexual cycles had been synchronized. Transfers of a two-cell uvum and an eight-cell one resulted in successful production of normal kids. In another experiment. ova were stored in a mixture of equal amounts of goat blood seruru and Ringer solution for varying periods of time. One transfer of four of such ova which had been stored for about 2-I hours was a success. and three normal kids were produced.

In 1970 Whitten [i0] detailed the nutrient requirements for the culture of preimplantation mouse embry os in vitro. He reported on the nature of the agents used arid specified pure water as the major component of the culture. He concluded that "all normal mouse embryos develop from the pronuclear stage to blastocysts when cultured in a simple chemically defined medium and a high proportion of these develop into normal mice if introduced into the uteri of pseudopregnant females. The rnedium should have an osmolarity - between 240 and 31(I milliosmols arid a pH of about 7.

2. Some oxygen should be present but the partial pressure should be less than 76 mm Hg. Calcium and bicarbonate ions are essential but there does not appear to be a requirement for amino nitrogen. Pyruvate is the essential energy source but care must be used as it inhibits development in higher concentrations."

Eggs of one species of large mammals have been implanted in the uterus of some smaller mammals for transportation purposes and then shipped long distances by air. The N were subsequently re-implanted into large animals. such as cattle. of the same species as the original embryo. These studies have -one as far as having preliminary commercial use for selective breeding.
One major concern in human experimentation is that while many basic principles are alike for all mammals. there are enough significant differences to make it difficult to equate progress in animal research with that in humans. For example. there is no question about in vitro fertilization having taken place in several species of animals. yet critical opinion is divided as to whether or not in vitro fertilization with subsequent development of multi-celled ova has actually been accomplished. The reports and photographic evidence presented by Steptoe and his associates [i7. 70] are quite convincing as are those of Hayashi and his colleagues [21]. But as Mastroianni [6] has indicated in his analysis of Hayashi's report the control oocytes that were not exposed to spermatozoa were indistinguishable from the cleaved ova resulting from incubation of spermatozoa. This observation weaken,; the claim for accomplishment of in vitro fertilization under Hayashi's conditions. The conclusions of Mastroianni's group in 1970 are as follows: "over the last three decades, in vitro fertilization of mammalian ova has sparked the interest of numerous investigators. Experimentation in the laboratory animal has contributed greatly to our understanding of the biology of the fertilization process and has paved the way for experimentation in the human species. Documentation of in vitro fertilization. by transfer to recipient mothers, with subsequent embryonal development or delivery of normal young. has been provided in the rabbit and mouse. In the human. visualization of two pronuclei and the sperm midpiece or tail within the ooplasm and cleavage to advanced stages have been reported. Recently. penetration of the zona by the spermatozoon has been documented with cinematography . The ultimate experiment - in vitro fertilization and cultures with successful transfer to the human uterus to produce offspring - has not as yet been carried out. Indeed. further information on the biological systems which would predictably result in the deliver-, of normal young should be a requisite for such experimentation. Eventually . techniques of in vitro fertilization could open the way for the evaluation of a variety of practical approaches to fertility regulation and the ultimate in planned parenthood."

In 1968, Chan g g [9] similarly commented m the status of ill, vitro fertilization of mammalian eggs and summarized the situation with respect to human eggs. He reported that "human ovarian eggs were cultured in vitro for 24 hours to induce maturation. and then were cultured further for 45 hours in the presence of human sperm by Rock and Menkin [36]. Altogether four of 138 eggs thus manipulated cleaved into two to three cells. whether or not these four- eggs were really fertilized is still uncertain in view of the probability of the occurrence of some parthenogenetic cleavage during culture. Moreover. Rock and Menkin [36] reported that "at the end of incubation period. the more normal of two specimens consisted of three well-defined. round. regular blastomeres. each of which contained a round. vesicular nucleus." In this later case. one would have expected one of the three blastomeres to be at the stage immediately before division. thus showing some evidence of mitotic chromosomes instead of a vesicular nucleus. Slrettles [15] reported the development of a human egg into morula after the incubation of an ovarian egg with human sperm and tubal rnucosa in vitro. He neither excluded the possibility of artificial activation of the egg in his procedure nor mentioned the probability of parthenogenetic cleavage in his paper. Judging from the published photograph. it seems more likely that the `blastomeres' were simply fragments within the- zona pellucida rather than those of a healthy morula. '

Edwards [14] reported that 80 per cent of nonfertilized, immature human oocytes recovered from women in various stages of the menstrual cycle could mature from the post-dictyate stages to the extrusion of the first polar body by culturing them for 36-43 hours. When human oocytes thus cultured were exposed to human sperm treated in various fashions. Edward. et al. (161. reported that four of 56 oocytes were possibly fertilized by washed sperrn. one of 1-f by sperm previously placed in the rabbit uterus. and two of 20 by sperms in contact with excised segment, of endosalpinx. Since their coo idence for fertilization was the presence of pronuclei. sperm tail. or the second polar bodv, whether or not these eggs were really penetrated or artificially activated is still in question. As shown in their photographs. the presence of one or two pronuclei without the identification of a sperm tail in the vitellus and that of polar bodies is not conv incing evidence of fertilization because of the possibility of spontaneous formation of pronuclei in the unfertilized W ''hat may be a spermatozoon in the perivitelline Space of one of their photographs does indicate. however. the possibility of sperm penetration through the zona pellucida. In this connection. Chang [l l] has reported that when oocy tes of rabbits were first cultured for 12 hours in serum. nuclear maturation (formation of the first polar body I was observed in the majority of the ovarian oocytes. Although a high percentage of these eggs were fertilized Mien transferred into the oviducts of mated rabbits. only two of d2 of such fertilized eggs were able to develop into normal y oung. Thus the acquisition of viable eggs and the possibility of their future development depends on the physiological stage of the cytoplasm of the egg as a whole. Nuclear maturation is not necessarily an exact indication of their potential fertility.

These sometimes conflicting reports represent the state of in vitro fertilization and embryo transplant. N\ Work in these areas in the human is limited l) \ many factors. one of the foremost of which is the "ethical" ' availability of human eggs. Obtaining ova requires informed consent of the subject. and often involves the use of hormonal agents to induce multiple ovulation. The latter carry a minimal risk. but patients must be so advised.

In our own studies. we have restricted ourselves to investigation of capacitatian in humans and to limited studies of animal in vitro fertilization.
Our capacitation experiments have attempted to determine the value of tetracycline in the labeling of sperm by ultra-violet fluorescence staining methods. Freshly ejaculated semen was compared to sperm cells obtained from washings of the uterine fundus by means of the washer. We have observed. in general. differences in the binding capacity of tetracycline-HCl in the fresh semen samples (blue-yellow acrosome and intermediate-piece fluorescence) in comparison with a lack of binding capacity (non- stainability) or a decrease of it (faint staining ) commonIv observed in fundal washings. These findings have not alway -s been consistent and we are not yet convinced that the tetracycline techniques will give us needed clues to capacitation. For this reason. we are trying other methods which we will publish soon.

In our laboratory work on in vitro fertilization in hoes. we have become increasingly aware of the need for more complex in vitro environmental instrumentation to produce normal fertilization and cell division to the blastoc yst stage. Present in vitro techniques for egg maturation are essentially "hanging drop" methods done in petri dishes. It is necessary to develop more sophisticated sys-tems providing continuous flow of media which will keep the environment unchanged or allow the inducement of intentional changes for research purposes.

Temperature control of the ripening or dividing egghas generally been fixed at an optimum of 37-38oC. Whether this is too arbitrary has not been supported by a great (lea] of experiments or careful monitoring. Culture media have been well studied from chemical. osmotic. and nutritional point,, of view. We believe that the Whit-ten solutions or the modern Ham's F10 or F12 are very close to providing an ideal environment.

In relation to the pO2 pCO2 balance- it is well known that the present knowledge is applicable to the initial stages of maturation and cell division. What does not seem clear is that such fixed values are applicable to later stages I 180-morula and blastocyst I. At these points more complex biochemical reactions and morphological differentiations take place. Oxygen requirements increase in geometrical progression: the blastocyst requires for nidation a much richer ox ygen supply. among other things. Perhaps this is a clue to the occurrence of increased malformations during fixed iii vitro conditions in advanced stages.

If we obtain these and other refinements for egg culture in a few Nears. it will then he essential to focus our attention on all of the very difficult steps required for blastocyst transfer to the human recipient.

1s we suggested early in this presentation. transplants of fertilized ova to anxious infertile females are at present la°v ond our technological capabilities. Problems to he solved include possible rejections. anomalies. and malformations. At the time of transfer it is likely that the development would be advanced with strong signs of tissue specificity.

The morula and blastocyst stages seem at present the optimum transferable. phases; transplantation attempted at earlier stages of cell division have usually ended in failure.

Laparotomies and laparoscopic procedures seem to be the logical routes for tribal egg deposition and fixation.

At present, there is no available literature concerning middle third tubal blastocyst implantation and blastocyst depositions around the fimbria. Development of surgical techniques and instruments are the most urgent needs for successful transfers, as well as better understanding of the hormonal synchronization for the female recipient.

The Tyler Clinic, Los Angeles, California 90024

The Editors of this journal would like to express their appreciation to the authors for their willingness to contribute to this symposium by correspondence on a controversial issue.

In vitro fertilization and blastocyst transfer may be a way in the future to overcome childlessness due to impairment o f the Fallopian tubes. The vagaries o f collect ing o f oocytes, o f in vitro fertilization, o f blastocyst culturing, and of artificial implantation in a hormonally synchronized uterus must be worked out. The possible hazards to the fetus as well as to the mother must be determined to be minimal. The risk-benefit analysis will be an important factor in future research considerations as well as in each individual case where, such an approach may be attempted.

The scientific community should observe carefully and objectively the developments in this field. We have to be concerned with overpopulation as physicians and as inhabitants of this world. We also have to be concerned with the individual patient seeking the help of a physician with respect to family size and child spacing as well as her suffering from childlessness. The infertile patient is entitled to the best possible treatment like any other patient.

If the present technology is not sufficiently developed and if there is still a lack of information on the possible hazards o f in vitro fertilization and blastocyst transfer, the application to human medicine should probably be postponed. This, however, should not prevent research in these aspects of reproductive biology. All possible efforts must be undertaken to provide more basic information for future applications in patients with tubular occlusion. We look forward to their benefit one day from the progress that has been made in reproductive medicine.

Comments and discussion remarks by readers of the Journal will be welcome.

G. F. B. Schumacher, M.D.