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C~ER 2 AILING IN 1= l~AC,lICE: AND SCIENCE BASE OF MEDICALLY ASSISTED WONTON this *lapser is }mused on papers pi at a workshop on Basic Science Formations of MA ly Assisted Corruption sponsor by the Institute of Medicine (IOM) and the Lard on Agriculture of me National Research Council. It was held A - USL 21-23, 1988 at the An sold and M Beckman Center ~ Irvine, California. For the workshop, the organizing committee developed a program that explored the recent advances ~ reproductive and develop mental biology that apply to medically misted conception. this chapter represents a report of science topics selected by the committee. It is therefore not a comprehensive review of recent advances in reproductive research. The workshop also initiated an interchange of ideas among those involved in patient-related clinical practice, animal IVFET, and those working in basic research as it applies to humans and other animals. Thus, this chapter indicates research areas that promise improvements in the practice of rVFET. First are some developments in human and animal IVFET; subsequently, the processes and recent advances relating to gametogenes~s, fertilization, preimplantation development, and implantation are disruccPl. This chapter summarizes each talk given at the workshop. The full papers contributed by each author are found in Apperdix A. Developments in Human In Vitro Fertilization E1] Eastern Virginia Medical School, one of the premier centers for the clinical practice of in vitro fertilization, has a pregnant rate of 18.3 percent, bash on the Or of arcs used to stimulate the ovaries with either follicle stimulating hormone (ESH) or human n~a,~1 gonadotropin art, and a pregnancy late of 37.6 percent he on the ~ Or of patients. Both patient age and the cause of female infertility have an effect on the oubccme of in vitro fertilization. For women infertile because of tubal ligations or endometriosis, the rate of viable pregnancies obtained by [VFET at Eastern Virginia is 16.4 percent. Although the pregnancy rate did not vary according to whether infertility was due to tnhun ligation or endometrios~s, age has an effect. After the age of 40, it Ones Ore c3iffiadt to stimulate the ovaries to price mature Is. Because Rupees often seek in vitro fertilization to mrercc~ne mane infertility, the evaluation of semen is important. Arrantly, this evaluation is descriptive and relatively incise. Factors sup as number of ~ m, Epenn motility, arm ger ~ 1 shape of sperm ~ ntsfe.g. head Shape and tad) shape) are important variables in achieving pregnancy. For example, if 14 percent of the sperm are "normal," and there are 50~000 In per tic oentimeter of ejaculate, in - 25 -

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vitro fertilization prig: arming pr~ies in 30 Perot of all attests. However, eve if there are 100,000 On per cubic centimeter, but only 4 pendant are Normal the Firs pro rate dog; greatly. Abnormal sunk often fail to fertilize an egg became of their inability to penetrate the e~'s protective omrerir~, the zeta pellucida, therefore workers have tried a t~niq~e ~ as "zone driller' Owlish holes are prig in the zone pellucida to permit direct an: of sin to the he plasm membrane. Bus far, this t~niq~e has not been successful. Archer technique, hen as zone ~?littir~, has }>Ben ~ at leery University, Atlanta. E'y this t~niq~e, the zone pellucida is Split hani~al ly. E~na~ies have been reports by use of this method. Basic science questions new to be at In order to Event better marks for normal art a~r~1 seem art to iT~prave the perforate of sham in IVIES. ~y~preservation (freezing) of in vitro fertilized ~ryos1 is another new ark promising technique that will be discuss in greater detail later In this Copter. If embryos are frozen for later use, the stage of Heir develc~xt at Be time they are placed In the uterus can be mashed with the stage of the uterine wall Arid, increasing the likelihood of a su~=cful pro. Before cryo preservation bare an option, Libby - C had either to be plan ~ He up or A. In order to avoid the ethical dilemma of Eat to do with excess embryos, Ore than the optimal ~ er of embryos were safeties transfer Eta the uterus. By allowing the preservation of embryos for later use, He technique of cryopreservation rues not only the Lance of multiple pregnancies, but also the Or of tog a ~n's ovaries night be subjects to hormonal stimulation to produce opiates for additional ads of Inter. He use of cry~preservation has item In ideal rate of pregnancy at the Eastern Virginia clinic. Neverthel—a, Eric science relearn is need to ~ the nary parameters for sulfur cry~preservation ark the possible deleterious effects of freezing on the embryo. In cases of ovarian failure, failure of in vitro fertilization, poor quality of eggs, genetic abnormality, or inam-~=ible c varies, the only option available to women wanting to bear a child is to use eggs freon a donor to perform in vitro fertilization with the husband's sperm. m e donated eggs most often come from IVFET patients who have received hormonal stimulation and produced more eggs than necessary for their own use. Scmetimes, however, women who are to undergo a tubal ligation agree to ovarian stimulation before the surgery so that eggs can be harvested simultaneously. mese eggs are then donated for in vitro fertilization and transfer. Donors are phenogypically matched with recipients and are screened for psychological previews and infectious diseases. Patients who 1 me t£=m embryo in this cog often refers to In vitro fertilized eggs that developed to twos to Farrell staged embryos. Alff~a~h there are Tree technical ly precise terms for variants early stages of de~rel~rent, the more precise term`; are used here only when the distinctions are important to the Carets under discussion. — 26 —

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conceive through egg donation may need supplemental hormone therapy to replace the estrogen and progesterone normally released from the ovaries. In such a patient, however, the placenta produces detectable estrogen by week 6 of the pregnancy and progesterone by week 7. Fertile ized eggs from oocyte donation, if transferred to the uterus by day 17-19 of the recipient's cycle, resulted in a 36 percent pregnancy rate and 80 percent of these pregnancies going to term. One benefit of oocyte donation! in addition to overcoming the problems mentioned above, is that a Lime period of optimum implantation can be achieved such that the stage of the embryo and endometrium are synchrcrized. This technique has been found to Increase the success rate of IVFET. (food halite ryes are critically instant to the sumacs of IVFET. Experience at many clinics shows that a number of factors determine the quality of an embryo. Some important factors include the way ovaries are stimulated with hormone supplements and the synchrony between the age of the embryo and the endometrial stage. Mbre research is needed to understand fully these factors and to establish unambiguous and unbiased criteria with which to distinguish poor quality and good quality embryos. RP-~C~ fertilization is more likely with mature oocytes than immature ones, research to learn how to recruit a more synchronous population of follicles is important. Such follicles produce a more sync hrono~c population of of capable of responding to the maturational stimuli. Failing the recruitment of mature oocybes, research is needed to develop reliable methods of maturing oocybes in vitro. All of these areas, if improved by I knowledge of the cell biology of early reproductive events, should greatly increase the ability to identify couples with high prnh~hility of sucks== and may increase the Cc rat== of IVFET in human clinical practice. Developments ~ Assisted Conception in Food-Pro*ucing Animals [2] There are critical differences between the gnats of -misted conception in animals and human beings. In human beings, the gall ~ to increase the reproductive ability of these with impaired fertility or, perhaps in the future, to avoid the transmitted of genetic disorders. In contrast, the goal of -existed conception in food-producing animals often is to increase the yield of milk or meat. Because the ethical barriers are lower and the financial stakes higher, a number of advanced technologies are available for use ~ animals that are not available in the clinical practice of human [VFET. Techniques in commercial or research lace include artificial Insemination, superovulation, embryo transfer, freezing of embryo, sexing of embryos, multiplication of embryos by bisection and cloning, ~ vitro fertilization, and the modification of embryos by gene transfer. All western European dairy cows and 60 percent of U.S. dairy cows are impregnated by artificial insemination (AI). Al allows bulls, affected for their franc mission of bilk producing genes, to achieve up to 500 inseminations frml, One ejaculate. Thirty Yeats of AI has helped to double the milk production of each cow.

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Arx~ t~niqwe ,~ widely in humans as well ~~ in other animals is a~latic~n, which irT`rolves ovarian stimulation by he administration of hormone suppler;. In bath anions and horn, Elation is uppr~ictable ark often ~sful. there has been son Us when came am given priming Be; of follicle sti~ating hormone (=H), ark Den highly purified forms of FSH am use, hat the reasons for these effects are ~lea'. In Tuition, studies of cattle and other specie; have fat that the cxx~; pried by Formulation safeties exhibit abnormal c~aracteristic:s. Beat urx]~tar~ of hormonal cycles arx] the effects of hormxes on the cell biology of merit oat; whim likely have dint abdication to the solution of scare of these p~lelmse the process of embryo transfer In cattle starts with Ovulation of a ax', ark subs~uerrt mating to a desirable Hal. He resultant erribryos are c:ollec~ Surgically and transfers to anther cow whose estrous cycle has ~ syr~zed with that of ache donor occur. Although the sat rate with embryo transfer Is r ~ table (a ~ ut 60 Entrant of transfers result in pregnancy), the technique has not produced great ~ in ~ Ik or meat production. The production of many ide ~ ira copies of embryos from cattle with highly desirable traits may improve this situation. For this reason, embryo freezing and embryo multiplication procedures are receiving a great de~1 of attention in the commercial breeding of cattle. Embryo freezing, in concert with embryo transfer, has been generally successful in terms of pregnancy rater. Freezing embryos allows for the s borage of rare breeds and preservation of a cattle surplus. However, embryo multiplication has greater potential for the production of large n ~ s of highly desirable cattle. TWO methods of embryo multiplication are used--embryo bisection and nuclear transfer. Embryo bisection is performed at a very early embryonic stage and yields at least two ~11 masses which are genetics ly identical . me bishop embryos can Aches be transferred for norm pro to a recipient oar. mere is a species specific limit on hcm many bisections of a given cell ~ can be done without compromising viability; with cattle, the maxim yield is four embryos free one. In n~ucl-=r transfer, a blasters (embryonic cell), or its nucleus, fray a valuable embryo is placed into an ~ e from which the nucleus has been removed. The transferred embryo nucleus promotes development of a multicellular mass that can be used to mate a number of copies or cranes. There ~ a great deal of interest in this method both in academic and commercial research. Since these techniques allow more precise selection of desirable traits, both have the pabential to effect rapid changes ~ the prevalence of ads with those traits. While the techniques divest above are frequently and s~ccfully used, other methods of assist repression are also being investigated. One of the newer approaches is embryo sexing. Sting of embryos; is of particular i~portarx~ to the dairy icy singe only female offspring are needed] for mink production. Sexing is done by three ethic. The - 28

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first is kary~ping of embryos by bisecting then and using one half for ~r~genetic analysis. the thief did of this method are Damage that often oomurs to the ~~ and ache variable reliability of ~ kary~pic identification of sex. The sad ~~ drays antibcxlies to Specific antigens oar the ~hryo6 to identify the male embryos. He attract of this i~ologic=1 metha] is greater than that of Typing and may bed Vilely use, did on ache availability of antisera. Finally, ~ hybridization Is have bed adapt fan Ear biology to sex embryos }fly labeling specific parts of the male Y Dad Stains by embryo bict?sy. Were are Capella reasons to develop are fully IVFET pry in food-pr~ucing animals. Such techniques pride a large number of Embryos for transfer, ray, and embryo multiplication. However, as in human WFEr~ ~ success of in vitro fertilization and embryo transfer In - He cattle industry suffers from a lack of basic krxowI~ge of the ~~] biology arm biochemistry of early gamete maturation ad fertilization. ng the arms in Ail s lapin is he maturation. As in humans, Iv~r in cattle is Are fur if it begins with mature rather than ilr~nature dates;. me biochemistry of sperm capacitation arm entry into the eggs also reds to be better dud. A nod of cats have been identified that ad nectary for these presses, but precise identification of the role each *him?] play ~ fertilization is for further progress. Finally, being able to maintain the grad of an embryo in Suture for a longer period than is now possible weld be helpful. Such an ability w ~ d ~ se the ~ anc ~ of a favorable match between the embryos c and endometrial stager. Gene transfer, if developed further, also shows potential for enhancing the productivity of cattle. With the development of techniques by which genes can be microinjected into embryos, the ability to alter the phenogypic characteristics of food-pro~ucing animals has become a possibility. In an early castration of transgenic technology, world; injected Ruse eggs with the gene for human growth hormone. me rating mad grew to ad Rice the size of nonnal Moe. such teleology cold be used in cattle to alter the genes for skeletal muscle in order to prepuce higher quality meat prefects, or to alter genes in such a way that biologically active syntax wed be secreted ~ milk. mere are many areas in which the results of basic Beard Ward further the practice of a ~ isbed conception and embryo transfer in both human beings and over animals. these irxlude ornate maturation, Sperm morphology, the biochemistry of fertilization, cryopreservation, ovarian stimulation, and molecular genetics. In addition, topics such as membrane biochemistry, hormonal control of testicular and ovarian function, gene expression In early development, and the cell biology of implantation are identified as areas of exploration that would make major contributions to the sue of human and animal [VFET. — 29 —

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His ~ Awes Otis of G~tog~;~ AL ~zvirulation t3] Comparative rearm con ovarian starvation of non-h~nran primal; and dc~restic animals such as cattle, horn;, shop, arm pigs raises Sting issues. Scam work highlights He diffen~es between Be normal estrous cycles of animals sum as cattle, arm the cycles of human arm r~on-hmnan primate. Yet there are promo that are On Hang species In the station of follia~lar develc~ent arm the in~ian of mature oceans. Ovarian stipulation arm ~wulation are exag~s interverltions into a highly regulate ~ysiologi~1 press. He rang physiology is not always racily manipulated. Develc ping strategic to cir ~ event the clifferenoes between nor ~ arx] in ~ ovulation Is a major goal of research proposed for this area. Non-Human Primates Non-human primates exhibit a menstrual cycle that closely approximate that of humans. In monkeys, the hormonal events of the cycle include a geometric increase in serum estradiol, mldcycle surges of luteinizing hormone (LH), follicle stimulating hormone (FSH), and a later increase in serum progesterone after EH and [~H decrease In concentration. These hormone levels are precisely synchronized in the normal cycle and lead to the development of one follicle, the dominant follicle, from which cvulation occurs. One of the purposes of superovulation is to encourage more than one follicle to develop fully, thereby producing multiple oocytes for in vitro fertilization. In monkeys, one of four chemicals is administered in combination with human chorionic gonadotropin to stimulate ovulation. these are human mencQa~sa~ gonadotropin, human FSH, pregnant mare's serum gonadotropin, or porcine FSH. However, monkeys sometimes produce antibodies that block the activity of hormones obtained flown cipher species activity, and clarion stimulation fails. Incus, them is a need to develop stores of monkey hormones deco be used for ovarian starvation in this species. the rinse to the a~ninistrution of hordes ~ stim~a~ce oration ~ also not uniform in non-in ~ an primates. In g ~ ral, estradiol levels are higher than in a natural cycle, the EH surge is lacking, EH concentration remains high over a long period, FSH is low, and progesterone remains high in concentration. However, in some subjects the ~ is a premature, spontanecus EH surge that is nck synchronized with fo1licular maturation; this surge StCp6 the follicles and oocytes fern maturing. Even if such an EH surge does not occur, there ~ a wide range of responses to gonadotropin administration. It is impossible to predict the response of any individual animal. me EH surge and the hetercgenous responses, however, can often be prevented by additional administration of gonadotropin rehearing hormone. Another difficulty often observed with superovulation protocols is an overstimulation of prolact~n secretion. These Repartees from the normal hormonal levels raise important questions relating to the efficacy and urn effects of ovarian stimulation. - 30 -

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Apiece the problems of ovarian sti~ati~ ~ nca~-h~nan primate, reasonable so ret m; have Ben achieved. Nevertheless, ~ are additional barriers to progress ~ this field. ~ Sony of In primate is soberly limit - . In ~;tioa~, the animals often c~ be Are Can once for ovarian stimulation because of Me iTr~ogica~ r~ to human, I, and porcine ~a~ins usual in the stagnation I. Finally, As must be zippered fmn ~ }fly either I~t~y or Party, surgical Unit; that are reprice by ethical cor~siderations arm formal legal oc~ns~aints ~ a l~ Her of repetitions per animal. Although these prowls of avaiiabilit~r, ~se, arm Be lack of no~h~nan gona~r~ins are more difficult to avers than t~ebiologi=~1 pr~bl~ofaverridi~normal~ysiology, Inns questions a}x~t ~ e basic biological pa ~ sees Robin Ha ~ . Resolution of the problems a-=c^~iated with the use of non-human primates in this type of research could be facilitated by an increased ability to augment natural hormone and gonadotropin release, by improved resolution of ultrasound imaging to identify mature oocytes and guide their collection by non-surgimal means, and by the development of cell lines that cculd produce larger quantities of non-human primate gQns~ctro pies. Finally, it is important to note that many of the same questions reman unanswered regarding human ovarian stimulation and that particular non-human primate species serve as the best mcde] for human reprc~uctive physiology. Domestic Animals Extensive ~~.c~ of artificial insemination in cattle has ~ ease d the genetic contribution of desirable huts to the overall supply of cattle. However, there is little likelihood that the Hirable characteristics of fen ales will be further is tar augmenting the male genetic contributions. Ovulation, on the ocher harm, Dined with embryo freezing arx] embryo transfer, has the potential to irx:rease directly the germ pool of desirable female traits by increasing the ember of offspring fray a Valerie cow. There are a nor of different; beaten non-human prima`; and clarestic anneals ~ the ways ovarian stimulation is a ~ Cliched. Sare of these differs arise fmn differences in the reprc~uctive cycles of the two grcup6. In dairy and beef cattle, stimulation is accomplished with the administration of either pregnant mare's serum gonadakro pin (PMSG) or porcine FSH. m e latter is preferred to FUG because FUG has a long half-life and often results in asynchronous ovulations. Porcine FSH is nct without problems either since it is contami~ by variably high levels of luteinizing hormone. Dean dhorio~c gonac~c~rop~n, routinely beck ~ human primate, is not USA ~ the Facial practice of ovarian stimulation of cattle. ~ oont~1 the variability arm lack of prExlictabilit~r in the estrous cycle; of came, current propels often include an injection of a Specific type of pr=;taglarxlin (PGF-2 alpha). this treatment caress the reg~sicn of the corers lutes, thereby artificially restarting the cycle. The sum rate of Cation ~ came has been variable. this is caused by a moper of factors including seasonal variability, breed differences, dose and timing of gonadotropin administration, and history - 31 -

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of previous formulations. Sew diff~s contribute ~ ctiffi~ Mortaring st~ies performed on different bows and, at tom, in Paring sties In we same breeds, Ott ~ with varied nines or in animals of varying age. It has been fat that, even if all the factors forcible are bled, ~ Swains a great Anal of friability In the success rate of ~r~ulati~. A number of stra~gi~c ~ Ore the gun rate are Firstly being tried. One is an attest to purify porcine FSH preparations to exclude the contaminating IlI ark, further, to detennire the FSH to IlI ratios that are bib for su~cctul Formulation. Ark strategy Is to try to ruralize R4SG with antibodies so that the complications bat arise fen ~ lord half-life of EME;G can be prevent - . Rf~ the lack of normal acordination of _ ~ a ~ endocrine egrets is a Cain charred in ~r~vLLLatlonl bravest lgators are also tryir~ to define He sources of ~py art develop ways; of normalizing a Ordinal series of biolc~ical events. Superovulation In c3c~ic animus other than cable also has had mixed results. ~ goats, the technique has been tr~c~usly ~ccful. Superovulation of sheep has also been largely su~mcful, art a few irritative strategies have been allied in this species. Sheep are seasonal brewers and attends have been made, with initial =~==, to induce ovulation in this species during tins Pen they are normally arts. In order to bypass the use of exogenous gonadotr~ins, workers; have er~ployec] antibodies to inhibin In s;h~?. In is a Stan made in an ovarian fold icle that suppresses FSH secretion. me blocking of ir~ibin with antibodies raises ~1 levels, thereby increasing the rntura1 stimulus for follicular develc~rent. Antibodies against steroids have also been tried in ~h-~-r to achieve the same end. In horses, Ovulation has proven to be difficult, and to require no~tar~ard protocols. me period of esteems or "heat" in mares is '~`c'~_1 1 or 1~ :~ bran.; at = ~1~1, .~ - ~~ .~_~_. . In addition, there are preps p ysiologim~l ~ nines In mare to prevent the develc ~ nt of grins. mese two aspects of e ~ reproductive physiology greatly complicate the sur~=c~ful application of ovarian striation techniques. Also of critical importance is the fact that PMSG, even in high doses, does not adequately stimulate the development of follicles in mares. Unlike other animals, where PMSG acts as both FSH and EH receptors, In mares PMSG has only AH activity. Some success has been achieved in mares with administration of porcine FSH, especially if combined with human chorionic gonadotropin. In general, however, progress in so superovulation in horses lags well behind that in other domestic animals, ewnent for the pig. me my limited of all dial abdications of s~run~lation in animals has been In the pig. Although possible, the technique does net confer maw of ache advantages that it holds for other species. One ran is that pigs deliver liters as q~ to one offspring at a tin - . therefore, the need to Pease He offspring foxy one individual, while helpful in certain cir~ta~es, Rustic animals. Never~c, Ovulation arx] embryo transfer may — 32 — __ , _ . is net as cc~maellina in Dices as in other

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be able to lower the frequency of din by ~ sing the number of offspring f`=u di=~'=-free or disease-resistant populations. Mbre frequent reasons for the use of embryo ~ fer in pigs are to obtain sew-free embryos f ~ u infected pigs and to 1ntroduoe new genetic material into specific pathcgen-free herds. Thus, further research into the normal reproductive physiology of pigs will be useful. Tb improve rVFET in domestic animals as weJ1 as in human and nonrhuman primates, more needs to be known about the normal regulatory events of ovulation, including the physiology and biochemistry of the development of a dominant follicle. Improved ultrasonography he= potential for increasing the I of superovulation in all species. As with parleys, developing ways to bypass the use of exogenous arc like MEG and porcine FSH weld be of enough ,~ in domestic animals. One way of doing this may }'e by blodki~ ir~ibin as has been done in sleep. In order to imps w e the mY~~=c= rate of assisted c ~ oaption In animals, many of these rv~Y~mch areas must, nevertheless' be pursued in each species separately with realization of the inter-species differences. It is not always possible to predict when the results obtained from one species can contribute knowledge applicable to other species. Biology and Maturation [43 This section examines three topics relating to the biology and maturation of oocybes: 1) The sway of membrane biochemistry, which has relevance to the complex membrane interactions that occur at fertili- zation; 2) the study of molecules that are important in maturation events, Is also important to understanding the possible reasons for failure in IV BET; arx] 3) the biological are physical pr ~ erties of eggs are embryos that are affected by preservation techniques such as freezing, known as cryqpreservation. Membrane Biochemistry The membranes of all cells of the body share certa ~ standard structural characteristics. The basis of a ~~l membrane is wiled a bilayer, which is a two-layered collection of phospholipid molecules (lipids containing phosphorus). Phospholipid yes are polar ~ that each has one end that is stable in water (hydrcphilic), and one end that repels waver (hydrcph~bic). In a bilayer, then, the lipid molecules line up so that the by ~ Cubic ends meet each other on the inside of the bilayer. The hydrophilic ends are thus oriented on the outside of the bilayer such that one group faces the intracellular space are the ether far the extracellu~ar spar. Sub an arrangement Mars sense when ore oc~nsider:; that bath the Neoplasm of a cell arrt the extracellular span are ~1 largely of water. It is, in fact, a quite natural rinse of lipids to form bilayers in an aqueous vironr~nt; such a process Berlin soap bubble arm oil droplet formations. 2 Embryos can be rinsed free of pathogens or tread with enzyme to destroy pathogens Eking embryos the safest method of Ovid germ plamn win t pathogens around the world. — 33 —

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Steins are part of Me membrane also. One proteins E - n Off thickress of Me m3ubrare and act as ~hilic Carts for ions or participate ~ Alar signal tr~tion Aim. Other proteins are partly erred in the lipid bilayer arm partly exposed to the extrar~llular Laos. Atta~ to sew proteins are Cog, often br~i~, chains of carbohydrate lies Cat also etch into the excra~llular space. Finally, large e~ra~1lular proteins often associate ~= or stick to the Nat Al parts of the carbohydrate molecules. Its, the ~11 membrane, ever In its scarab fond, is a complex structure made up of differ An; of molecules array In regions that each exhibit - ^ - - ~_ ' _~ _- - If: ~~ ~~ ~_: =~ ~ : ~~: ~~ ~¢ ~~ ~1 ~1 ~ 9 ~;1^ 1 ,pL~ ~1~. 110: i~—~1= AL—~ "~D Vet "~`w`~ `~ - ~ · ire since He variants Leaves are fertile within ache bilayer. these interactions orderly 03 Rally immense range of biological r~ of c - 1 is including ~1 1~1 ir~eractioa=, r~r~ia~ signal premising, arm t~brane transport of nutrients or t=XinD- Reproductive events In which Crane biochemistry is of particular i~ortarx~e include sperm capacitation arm fusion of serum arm egg membranes for fertilization. Relic assays of membrane biochemistry are Nat often sixties using membranes fmn owls such as red blood Ells or Bran manufacture in the laboratory, called likes. Such Rare are simpler Man the Umbra fmn eggs or sperm ark, therefore, easier to sky. Hirer, data Caine freon t:h~ Gel Ares nest be excrapola~ ~ Us arm sperm with Goat caution. Lillian Locke Maturation . . . In a nor foil icle there is Fornication between the As and me ~r~g reverse cells, called gra~osa ~1 IS, through specialized junctions (gap junctions) threw which smal l moleall.es Franc fold ore roll to ark. Developing A; undergo a type of ~11 division in which only half the normal nor of ~nc; are retained. This pr~c~;s is kncr~7n as Kiosks and he a Enter of stages. Until just before maturation, however, the growing oomph; remain In a sea te of susp ~ ed or arrests meiosis. It is th ~ ht that the granulosa cells help to ma Stain this arrest of meiosis by producing substances that enter the oocyte through the gap junctions. It ~ also possible that substances that induce final maturation of the oocybes are prod urea in the surrcundinq cells and are transmitted to the oocytes in the same manner. Since the ability to mature oocybes An vitro would be of enormous value In IVIES, the u~rsta~q of the s~s that either maintain or abolish meiotic arrest Is particularly desirable. It Is important to note that many of the studies satirized In this section were done with mouse 00~5 and these data were cc~panad to those Stained from other Spinals incl~i~ra~its and rats. Ir~vestigators have also studied frog oocytes, but the mouse is the my an animal meek. Evenh,~1ly, of Carrie, ~xr~= critical - Stints will have to be replicated using oocybes frill human or nonrhuman primates. Several substances are thought to maintain meiatic arrest. One, cyclic adenosine monophosphate (cyclic AMP), is a molecule present ubiquitously In the body. Another, hypaxanthine, is common in the body - 34 -

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are Mulcts to a gasp of Yes 1~ as Enshrines. Guarx:6ine, art Enshrine, is also a potent inhibitor of Meiosis arxl the purine, Antoine, he been stun to aunt flee activity of hy~hine. E,y Dominic He effects of sane of Due Stan an eat Our, a hypes he arisen whip suggests that hy~chire are aver prim; may impose ache Intel of c~yclic~, which then ir~ibits~ios~s. Fire Drub, f~ ~ - n Be ampules of the cyclic AN pathway ithibitir~ meiosis, may eluc:idate ways In which i; can be maintain In Civic abut In vitro. Bile knee rqa~i~ the ithibiti~ of oboe maturation Is important, kr~ri~ hcr~v to macro i; in ~,l~re would have many applications that are describe later. ~ viva, Ye ma~rati~ ~ prig by ~ surge of 111 i~t-1y prior to Ablation. she Beanie of action of Ill Is uncial, however. Cue might assure ~at, if Chic AL mains Idiotic arctic, Awe IN sat cause; a Lease In Chic AL In ~ cue or ~ ~rr~ir~ nurse delis. It he been On ~ viva that administration of It in combination with human ~a~-~1 gonaclotrnpin rout cyclic ~ in the ovate, but not in the s~rm~di~ curse cells. His effect is puzzling since the gap jurx~tions between the ox; are the ocher Plus still appear to be functional, so cyclic AT dhalld Ego through these junctions Oily. An alternative hypothesis is that Ill acts icily by causing scale maburation-inducing Stag to be pr~x3u~ in the ~rr~ir~ Bus that duress the cyclic AD in the ~e. Since an Crease in ir~cra~lular calcium has been shun to be Naiad In sanatic cell division, it ~ bible that a ma~tion-ir~ucing ~ curate in the Cues by caroling an Case in ~tra~lular Cain. me sear for these suntan art their anti of action Is a major area for fug rehear=. she stay of o ~ ce ma Oration has led to etch ~ ]y sun ~ =ful methods for maturing mouse oocytes in vitro. Such work has shown that the in vitro culture of oocytes and their surrounding ~P1IS can be accomplished in ways that allow subsequent fertilization, implantation, and delivery of normal young to ~ Jr. Sur--==fu] in vitro fertilization of oocyte_ from other species, however, depends upon continued basic research on oocyte nutrition and met~holism and on the factors or substances that control differentiation and maturation. m e potential applications of an increased ability to mature oocybes in vitro are numerous. As disr~=c-d earlier, such an ability would be of enormous value in the production of agria naturally important animals. m ese techniques also have important applications in attempts to preserve endangered species. For example, it had been demonstrator that oocybes removed from mice up to six hours after Beth can be matured in vitro (Lazarus effect). Yet another application can be found in basic science research into genetic dic.~c c through the ,~== of transgonic ~ ce. Such mice are pro*uoe1 by introducing known gene fragments into the gonome of a mouse. These gene fragments have been inserted by m~crv~njection into eggs. Alternatively, the fragments, incorporated into a ha ~1~ virus, have been Injected into neonatal female mice. In this way, such fragments are incorporated into the DNA of developing oocytes in these females. It - 35 -

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question mUL=t be known. Some of the sequences for human genetic dicpacpc are known, especially the sex-linked i... The problem, however, i_ that many in situ probes are linked to the Y sex chrarosome. Thi_ causes an unacceptable rate of false negative readings. A procedure with greater potential is to look at the defective gene products/ like hemoglobins as an indication of genetic blood d*co~=-c, or like hypaxanthine phosphoribosyl tran_fer~e tHPRI) as an indication of Lesch-Nyhan Hi.=P~=P. The last prerequisite is absolutely critical, and that i that the probes most a.==-== the activity of embryonic genes nek maternal genes. Studies ~ the mouse have suggested that the -embryonic gencme turns an between the 2- and 4-cel1 stage. Before this stage, intracellular proteins are determined by RNA which was stored in the oocybe. Investigators at the Medical Research Council Unit of Mammalian Deveiapment in Landon have developed a m~croassay for the presence of HART On embryos that, applied in mousse embryos, has begun to answer some of the questions about the time of appearance of embryonic gene activity. HpRr is an enzyme that is lacking in children born with the sex-linked Lesch-Nyhan dilate. The hallmark of the Use is self-mut~ ation and such children also exhibit strange motor behavior, are often mentally retarded, and usually die by the age of 10. Using the Acre essay for HPRr, workers have found that the enzyme increases in concentration in 8-cel1 mouse embryos suggesting that the embryonic genes have become active. So, it seems fairly clear that, in the mouse, gene activity begins at this point. However, before pre~mplantaticn diagnoses can be achieved in humans, one must know the timing of human embryonic gene activity. In Great Britain, research on human embryos is allowed under the con L`vl of the Voluntary Licensing Authority. In the Cambridge University clinic, all patients are asked to donate their excess eggs and embryos for research Is. Of 300 patients, only two refuse permission for the research, an] thick two were set 1 afforded all the clinical services available. I Investigators at this clinic have thus con ~ EN a number of stNdies on human embryos. In a series of experiments, radioactive methionine was added to the couture media of cultured embryos at various stages of development. Methionine is taken up by cells and incorporated into newly synthesized proteins and so is a qualitative measure of protein synthesis. Researchers found new proteins that contained the radioactively tagged methionine were synthesized for the first time between at the 4- and 8-cel1 stage. In another experiment, when chemicals, which blocked transcription of messenger RN\, were added to the culture medium, they had no effect on unfertilized oocytes or early embryos and development pro seedy normally. However the blockade of RNA, or protein synthesist from the 4-cel1 stage caused the embryos to stop developing. While the results suggested that embryonic gene activity! which directs synthesis of new proteins, is important at the 4- to 8-cel1 transition period, the data are complicated by the fact that embryos have a tendency in vitro to stop development spontaneously at this stage. m ere is, however, further support for the idea that the embryonic genes are active at this stage. Since earlier experiments had looked at the - 55 -

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pattern of proteins present in early awl late Embryos and faced that the pattern chased quip clearly, the protein patterns were Irk with and without Me blockade of I_—r I. -these results ~trsted that, Men ~ synthesis was blocked, Me ct3a~ in protein patterT1 characteristic of Me 8~11 stage and beyond did rat War, but Me pants maim in the early state. Becky tile Objective of such remark was to develop p~i~plantation diagrx~tic techniques, Me pr~e of HI was also assayed Frau hen embryos. Unlike Me fifties ~ ~ mouse, there was a large variation in EM levels an human embryos am the massive rise ~ HEM at Me 8 cell stage Characteristic of the at of gene activity in Me Muse was not son in the human. lee Masons for these diffe~xs ark for di~ies between these data arx] that of others are not clear. It Weld be that EM is synopsized by the human Embryo later than in the noose embryo, or that the Hear Frau the maternal gee - ; ~ being broken dawn as rapidly as new HI is pry so that no net impose can bee seen. It is also possible that there are simply ur ~ rained cliff ~ ; in storage, culture conditions, or cipher variables. Whatever the reasons, the application of HART assays to preimplantation diagnosis of Lesch-Nyhan Syndrome in the human has been disappointing. The situation does point out, however, the dangers of extrapolating flown animal studies and undersoores the need to conduct some research with human embryos that, one ~ ise, would be wasted. Regulative Pobential of Micromanipulated Embryos Many of the classic studies on embryonic development involved manipulation of certain parts of animal embryos and subsequent analysis of the effects of that manipulation on find development. Such manipulations included red of certain Is (limb buds, for exile, to study regenerative capacity), transplantation or exchange of parts (as in Elisha ~ ing uE—r Spinal cord regions with lower Spinal chord to see if nerve outgrowth would follow the limb buds in the normal way), and grafting of parts of one embryo to another. Many new technologies allow m~cro-manipulation of embryos at the level of Calls and, sometimes, even individual molecules. It is clear that the developmental potential of cells gradually narrows as develcpment proceeds and that embryos have remarkable capabilities to readjust to disturbance. Yet little is known about what governs the point at which readjustment can no longer occur be the pot Partial of a given embryonic region Hal been irreversibly determir.cd. Further, it is expected that that point of determination will vary according to the specific region in question and according to the species involved. It he long been thought that prior to blastulatian an embryo is totipotent, meaning that no ~11 of the developing embryo is committed to any particular develop mental fate. In other wards, the minis are - 56 -

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undifferentiated and uncommitted. However, it is becoming evident that embryos of different species, even though exhibiting the same morphological state and the same number of ~Is, may differ ~ the degree of determination and potential of each nail. Such findings call into question the long-held assumptions of totipokentiality. since an embryonic ~1 1 is totipatent only if it can Revels into an entire organism, e~eri~nts con embryo Splitting can answer question r~i~ ye real potential of embryonic calls at different sees are in different secrecies. If a 2~l Encage embryo of a 1~ - ratory muse Is bisect, develc~tpr~normally. Unto the stage, individual blasts can be dish from an embryo and aggregated Inch art embryo fully. Ever, such 'related blast Erg: develop or organize into viable fetes- an there En. he findings In the rat are s;~nilar. In contrast, refit blast isolate Emu 4- and 8~11 stage embryos have been shown to be totipotent. Other experi~s have Emil the effects of bisecting Fuse embryos exactly in half. File 65 Event of half embryos sunrive f`~ the 2~l stage, anly about 45 pi survive fern later stages. me Exam at later so may not Sean that all the cells are toting; rather, it may mean that each half embryo contain the r~ui~ ~ and type of ~11 to Deplete develc~nent. Information cn Panic Lies Is derived by fen ex`~rin~s with sheep and cattle embryos. In sheep, isolated blasts meres have been shown to develop normally from embryos up to the 8-cel1 stage. However, for unknown reasons, only some blastomeres are capable of such development. One case was reported in which an 8-cel1 embryo was divided into four equal parts. Transfer of these split embryos into ewes resulted in the birth of fair lands. Sheep embryos, which have been halved, seen to survive well fen the 2 ael1 to blast stage. :Cn cattle, similar sum= has been achieved with Eatery arx] halved embryos fog. the 8 oe11 stage to the blast~yab stage. ~ to the ~se, Lip art cattle exhibit blas~ation at a later cleavage stage. Therefore, the n ~ of cells ~ her at blasb~ation for Sheep arx] cattle. It is possible that a higher total oe11 number may allow for greater flexibility embryonic adjustment to manipulations such as splitting. Beyond questions of immediate survival of micromanipulated embryos, there are other questions that have been addressed by investigators. Some studies have assessed the size of embryc6 after splitting and the birth weight of resulting offspring. While me study in mice found no differences in these mEasuremcots between control and halved embryos, others have reporter differences at various times of development. An examination was made of blasbocyst formation following 2-cel1 stage embryos that had been bisected. One half of the embryo was discarded and He other was cultured, returned to a 5~ize~ fee, or placed In an immature oviduct. Iho~;e placed into culture critic e~;:bited Ayes - 57 -

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<3evelc~rent. A hider Its of the half marry=; <3evelc~ nonnally men returr~ to a yr~ized fame or an in ature adrift. Sum fistic indicate that playgirl the split Embryos in vitro sashay Rises their c~velc~nt. However, it is possible that Be User of ted half efforts wee ~r~tilnat~ in Me in viAro ~itic~ns, since it Is Ire cliffiadt to recover arm identify Grated Oryx; fmm the USA; or Writs. Be sties f~ ~ Hat viability was oorrelat~ with cell er sum that the lacer the ~11 number achieved in the first few days after bisection, He lam He can es of sur~rival. Many of He half embryos; fails after implantation, irxlicating that ~ bisection did not rat in a failure to implant. One of He most clear suggestion Frau these sties is that ~ ~11 rnmixr at blas~atic~n owlish Augur'; at a particular time, irk of the cell Amber) ~ critical art It; blastlllation results in an embryo with a 1~ Beer of inns ~11 mass Ails. A reduction in the inner ~11 mass may then cause the Denise of scan half embryos. Is idea was tats directly by ark gram of skies that use diffe~ial stains to carfare the rumba of Ells in the Bar cell mass ~ me Gal namer of ~ is. Embryos with a lax ~1 1 Beer at blas~lation hac] a lacer inner ~11 mass ratio and exhibited a lower viability than embryos winch a higher cell Trim at blas~lation. these experiments further she that maintaining the half Oryx ~ viva, even in a nor'?regnant uterus, Gas the formation of the inner cell ~c=, resulting in a higher inner ~11 nsss ratio arx] higher viability than that achiever] in vitro. In a Sony =~ the lantern effects of Kayo splitting, genetically identical embryos Ore either bisected arc] immediately plasm it foster ~thers-or were left intact art plan into foster ok. He half erdiaryos were generally To C~cful am those born had a higher rate of neonatal mortality than did the controls. However, sane of ache neonatal Mortality ought have been I the half embryos were born fern smaller liters than He controls. small litters usually result in longer potencies, which are also associated with hither neonate] Gail rate=. ~i~n of pi~er~ypic c~cteristics of the negates born revealed no significant differs= between half am] whole By n measure such as the are at eve opening or Row curves. . . . . . . . _ _ thus, by birth, sex type of regulation has taken place. the stoics taken together argue strongly for ~ Lists of crisis points for half embryos ir2cludir~blastulationarx1 implantation. F=ther stay of the Gil of regulation in mi~nanipulated embryos net Ply can ~ to arc basic Repledge of Rive biology, but can also elucidate important rats for fur splitting of embryos in different species. :[nplantati<~n to] ~plantation, one of the Nat poorly urxierstood pros in r~pmductive biology, involves a Alex interaction bets the Kayo arm the Ells of the uterus. ~ are also significant Ties differences in the pro of implantation that can be generally divided 58

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into Irk types. Cane type of i—la~atian fat in rabid s ear led fusion; Was the type fat in rows is calls display:. Plantation in hens ark Mar primary is rolled intrusive and is the inportarTt to ~ for the pa of this report. we germs Mel for implantation in the hen begins with the altar of the blas~st to Me Dial wall. At this point, He alter layer of trim of the rye proliferate arx] differentiate into two types of Blast Ills. He Ills clcee to He Frye Bose ~tr~lasts and the cools ~ dint Fact with He ~530rium few with each ather to be He ~yr~tiotr~l~t. A ~ytium is a large, n~ltirn~cleated, mass of ~rtaplasm for }fly the fusion of arty strafe ~to~h~blastic cells into me. He syn~,tiatz~l~t acts to Be a path into the e~anetrial tissue ~at, ~ turn, allows the embryo to burro it He wall of He uterus. Scan Ells of the e~anetriurn are ir~ucc~ to Kilt] up large stores of nutrient Leaves, which are Ten released into He excracellular span (close ~ the erdbryo3 as the=- Ells degenerate. ~ charges in erx~anetrial cells can be visualized with a ~ of Deal Cliques ark are called the decided ~action. Eventually, the try plastic cells penetrate cheaply Oh into the uterine wall to Apace maternal blew veals. He iT~ceractions between the tr—cblast and these bloat vows results ache formation of the vascular supply of the placenta. The sty of implantation involves ache sears for and i~icification of factors conning uterine r~ivity are maternal reposition of pi. Me of them factors seem to be pried arx] relend by the His of the blas~cyst prior ~ implantation. me Bard of the biology of implantation has far-reac~i~ implications for human and nonhuman reproduction. Both in the natural reproductive process and in the practice of medically assisted caption, there is a huge gap between the Oar of sue awfully fertilized Ed ark the Faber of offspring bon,. A large part of this gap can be absented for by the 1~C of embryos at or AL the point of implantation. For Ample, in human IVY practice, 60 to 70 pennant of the eggs are fertilized, but only twenty to thirty percent of the embryos placed in the uterus result ~ an ongoing pregnancy. moreover, it has been estimate that, in couple without fertility problems and not practicing birth control, a prearm ogre; ~ e art of three Trust Holes in Nigh a fert;1izec] egg is present. In *seep art cattle, twenty to forty pen sent of the fertilized eggs do not survive and, in the pig, the Len rate is thirty to forty percent. early embryonic 1~c~ is common across species. While some early embryonic lace can be explained by factors such as heat stress, nutritional deficiencies, and genetic abnormalities, it has been proposed that much of this loss results fray three other possible candy-. First, the uterine environment is probably only narrowly Dive to implantation by an embryo. Seed, embryos may fail to signal Heir preserve and, consequently, fail to induce the nor hormonal and uterine changes nary to maintain pro. third, embryos may be rejected as a foreign body by the Therms imnn~ne sylvan. — 59 —

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ye idea that the uterus ally implantation ply user praise c~xlitions aft certainly underlies the primly dice els~e this chapter relating to By between the "cynic stage are the uterine stage. Iac:ik of Prey has bee Rated to be a problem in all species family. It has bee sin that if praise By cannot be achieved, it is Gully better to transfer embryos that are Are ~ than the uterine Nit. Tnis makes sense if ane residers that, foll~ir~ ovulation, the corpus lumen ~ the ovary begins to pry pr~ter~e, fillip acts to prepare the Us for pr~.If the corms lutes degenerates, the dreg in pmg~tem~ calicmc the uterirx3 lit ~ be ~ retire In ~tr~ticxl. A hormone sated by the therm, c~horionic g~adc~tr~in, prevent; the duration of the corpus luteum and, thus, maintains the pr~e suction. It is reasonable then that an embryo that Is trarmferr~ to a uterus Are Alvarez than the embryo may not prcx3~e High *,orionic Strain to be able ~ rescue the corms lumen few Gratis. there have been att~cs~to control the state of the uterine lining through the a~ninistration- of Us hormones. Attends to Vance the uterine errcriron~nt of pigs by a~ninistration of prone have been unsu~=cful. However, became pig embryos sate estrogen which also affect; the uterine lining, emus estrogen was tried. lhis Sarah worked, but only during a narrow time wire are, if given too early, was actually toxic to the embryos. Other rearm suggests that it Is too simplistic to assume a~inist~tion of one type of hormone Hula be sufficient to control ache uterine enrima~nt. Analysis of proteins synthesized are secreted by embryos indicat - : that there are a Or of different chemicals made by ethos that can affect the ~ erus or ache corpus lubeum. It is nevertheless probable that some of the embryo-produ~c~ proteins cause change_ ~ the secretions of uterine calls that am nenP=c~ry anchor supportive of an implanting embryo. A number of prokeins-are also secreted by endometrial malls, and much research has focused upon isolation of these proteins, analysis of their function_, and ~ pping the changing patterns of protein synthesis associated with implantation. One experimental strategy has keen to late] implantation sites in mouse uterus with a ~ye, pontamine blue. this ~ye, when injected into the veins of-a pregnant mouse, cards implantation site= to be colored blue without staining the rest of the endometrium. After this labeling, investigators can then remove the uterus and maintain explants of it in culture. Using precursors for protein synthesis that have been radioactively labeled, investigators con label the new proteins synthesized and compare the patron of proteins fran ~lantati~ and non-iT~plan~tion site. -this strategy he chain that me rate of protein accumulation in implantation site; is up to forty pent greater than aver site:. =dh of the grease is accamted for by Is ~ proteins destined to be secreted Frau erx~trial ~1 is. F~ work Ire the pattern of protein synthesis freon natural i~?lantaticn sit~- to those that were ~ animally Rued to look like implantation sites. Although there was an inazease in the synthesis of some proteins by the mechanical method, some proteins were not Increased. Such a finding - 60 -

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art for there being embryo preteen synods In the anetri~ at implantation silo. As will be discos" In a later part of this car, it is on that "oryx release Main factor arm prcteins bat directly affect the cells of the er~anetrium. At this point, beer, it is -fur to cxx~;i~3er saw of the proteins fat ~ be reload by erxia~trial calls. E~c~netrial pried 15 has ban imaged In home. This pried only present in the secretory phase arxl first trier of pro. me pro of this protein has bum ~ to charge ~ on the hormonal state, but so far no Fannie has ban ascribe ~ this preteen. Erxic~trial panted 14 has been fad In human and Muse er~aretria. this protein is identical In Strom to an I0F, whim ~ present in as sites of ~ body. en rapids, a pried Select utemglob~n has been shaven to be irrluc~ by pr~e and present In only prearm or In F~u~c~pr~y. Ihis preteen has a dried of fur~ic~ns incl~i~ an a~i-infla~natory action. Factors isolate freon the mouse include ppidermal girth factor arm a type of colony stimulating factor, both of which increase in rinse to progesterone. It is surmised that these, arxt probably Her, gram factors may furs tion to amtro1 the proliferation of the placenta. Studies of ~lametrial proteins ~ the pig are of special interest since this .~ cics does not e Exhibit the intrusive type of implantation. In fact, the maternal and embryonic blood supplies never come clod to each other in the pig as they do in humans and other primate=. Such a situation sets up potential problems in bringing nutrients to the developing embryo. Nutrients must be red from the endometrium and diffuse to the embryo. Proteins have been found in the pig that seem to help in this respect. For example, uteroferr~n is a p Stein that carries iron to the embryo. Another type of p Stein, which increases in response to progesterone, transports water insoluble molecules to the embryo. Thee are Called retinol-binding proteins. Two other proteins plasmin and tryps~n inhibitors protect the uterine cells Frau destruction by embryonic enzymes, and lysozyme protects against infection. In summary, many Classic of proteins are produced and secreted by endometrial cells in response to estrogen, progesterone, or embryo-pro~uced factors. While some of these p~V~ein-c serve nutritive functions in species in which the embryo either invade_ the endometrium late or not at all, other functions of these proteins are not clear. It Is probable that early embryonic In== is, in some cases, due to abnormal eXprpccian of uterine prcteinC. A poor quality environment for the embryo could result from either excessively low levels of necessary proteins or excessively high levels of proteins, which card be tactic to the embryo. TPCC meal has been ~ to eking the incite or ani~5 of ~nologi~1 rejection of embryos. The uterus Is no isola~ fmn ache inform Dyson, Medially In species In whim the maternal and fetal blood are hardly strati. An embryo is like a trar~lant~ organ, whi~h~stbe transplar~t~1to a site that is p~W~ - 61 -

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fmn ~ cells or an ingressive c3r~ ~st be given. In sane species, an i~plantir~ embryo CZ`~,,~-- what locilcs very ~ like an allergic inflammatory reactic~n ~ the Atrium, Cat is, dilatation of blood vowels, proliferation of blood capillaries, aTx] fluic] ablation. ~ addition, it Is McCann that ~~l surface antigen; Able of eliciting an ire reaction on the part of the mother are eventually present on embryos. me, ~ ~ why act embryos are scat rejoin ideological ly are contesting arm important areas of rag. It has been fag Hat embryos release Stage especially interferon. that. _ ~ _, _ _ _a~ _ _ _ . . . . . . . . . . 1n our tissues, act to suppress or mxhllate Me Gil act. therefore, it is possible Mat the embryo acts to control locally the in Sense of the Her by secretiall of inflating A. Aver, Bra rearm Is ~ before sub a Knin can be established. lie Obey of iT plantation in human beings is particularly difficult because there is no in vitro model available. Such a barrier has special importance becalms= of the wide species differences that exist in regard to implantation. Findings from other species cannot be assumed to be true for human beings. However, it is possible to ~ some aspects of human implantation by using in vitro methods recently developed. -One of these methods involves the isolation of cytotrophoblast cells. It has keen found that cytotrophoblast owls placed into culture under certain conditions will proliferate and differentiate into syncytioLrophoblast. Such studies have shown conclusively that sync ytiotrophoblast is derived from cytotroph~blasts. In a m~1bNre dish, this differentiation takes place in two steps. First, the troph~blast cells aggregate. Then they fuse to Bone a syncytimn. It is likely that this pro Is mediate by -adhesion Alleles or Cams, which have important roles in ~1 infractions during cievel~nent of the rearms system arm ather tissues. me CAMs are Prague by the tr~ablast cells because blockade of pr~ceirE(ynt: s prevents aberration of the cells. It Is also known that the process depends on Lain, since it d~ not aver in Are An catkin Lain. In contrast to the aggravation step, the Panic risible for fusion of the tr~ciblast are entirely Am. me Infractions of cage tr~oblastic cells with variants compor ~ s of ex ~aoellular matr ~ have also been s ~ ied. Excra~1lul matrix is simply the Intercellular space and its oc~Qanent molecules, which are usually synthesized by the surrounding cells and secreted into the extra~1lular space. m e exact composition of the matrix varies from tissue to tissue, but it is important in the uterus since interactions between it and the blastocyst occur during implantation. For trqphoblast aggregation and membrane fusion to occur, there must be serum proteins in the culture media an4/or the dishes mast be coated with extra~lular matrix proteins. It is Thought that the serum is required bec a11=r it contains matrix proteins, (e.g., fibronect~n). If these proteins are added, the serum is no longer requited. Collagen, fibronectin, and laminin are structural proteins that are plentiful in many extra~-llular spaces. — 62 -

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The importance of extra~-llular matrix was underscored by additional experiments that and endometrial explants to cc-culture the trcphoblast ~Is. This method~is more like the natural situation, singe the traphoblast differentiates into sync ytiakraphablast, as before, but now interacts with endometrial tin- m. These experiments showed that trophoblast from first trimesber-and term placentas bind to epith£1ial a-us of secretory en~ometrium. m e Otis also bind to cut surface= of the explants' areas where extra~-llular matrix was e ~ . After 24 to 48 hours, a zone of tissue recauais deve~qped~in the endonekrial explants where the sync ytiotrophoblast ~=cn~iated ~ th the.tic=~e. Mbreover, - . trophoblastic celis.-bind to and invade nests-of Burke endcoetrial gland cells. The trophoblastic cells dislodge the en~ometrial cools and penetrate beneath them in a pro-.-== resembling intrusive implantation. . These experiments suggest that the extra~1lular matrix always permits attachment and differentiation. of the sync ytictrophoblast. That fact has important implications in d;~-~^ce stabs= where the epithelium is eroded and, especially, when the lining of the fallopian tubes is eroded. Such static may cause implantation to occur ~ either 1es~= desirable sines or totally undesirable sites such as the fallopian turf=. The mechanisms for trcphoblast invasion of the endometrium are nak known. There are a variety of pr~teases (enzymes which break down proteins) that have been implicated, such as plasm mogen activator. In some strains of mice, blas*ocysts are less invasive of the endometrium apparently because they produce 1-== plasminogen activator. Cultured human trqphdblasts produce plasminogen activator (urokinase). Urokinase may degrade fibronectin and activate other enzymes (e.g., collagenase). It is possible that the action of prc teases like urokinase could be controlled by the presence of ~11 surface receptors for the enzyme, which localize its actions, and by specific plasminogen activator inhibitors,two of which are known to be generated by trc~phoblastic ~Is. This control would be important since the invasion of the endometrium must have an endpoint since embryos do not burrow all the way through the endometrial wall. It is expected that many of the biochemical interactions between the traphoblast and the endometrium Could occur through paracrine Mechanisms. Substances released by the syncytiotccphoblast and cytotrophablact could reach a high load concentration in areas of the en~ometrium. Sobstanocs known to have powerful paracrine effects in other tiC=~Fc may play a role in implantation including protein and steroid hormones. Substances red from bath the trophabl~=t and the endometrial opals, could account for the inductive and interdependent changes in both these tissues. Further research, however, Is clearly necessary to answer these questions. On the This of the above studies, investigators have proposed a working model for implantation in the human. The first step of this model holds that the troph~blast binds to specific endometrial ~1 l-adhesion molecules. After binding, the trcphabla-=t penetrates the endometrium and - 63 -

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attach to the extracellular matrix by Manic involvir~ pa ark controlled bar specific ir~ibitor';. Tnis Mel, then, form an Mate starting mint for further I. Kr~l~ge of the bi~istry is particularly deficient and OCR for page 25
E21INO:ESS PI ~ tactic ~ Dr. Zev me. 2. Spray of tale given by Dr. Neal First; also ~ paper by this author In fix A. 3. Spray of talk giver by Or. Robert F. William and Dr. Jerome Fortune; also see pat by these authors c A. 4. Seminary of talk given by Or. Ray H~rst~, lDr. John E\:3?ig, and Jar. Stanley Leibo. Drs. }~c~st~t's art E=ig's pal are included- In fix A. For Optional information on crycibiolc~y, see S.P. Deibo, Physiology: Elation of Aeolian Embryos. In Genetic E~gin~ring of Animus, J.W. Evans arc] A. Hollacnder (Felt.), Plen~ma fess, New York, 1985. Smnnary of talks given by Dr. William W. Wright and fir. James M. H~r~; also see pat bar these authors In fix A. 6. Gunnery of talk ~ Dr. Jonathan Van Blerkc~n; see paper by this author in Admix A. fink sully of talks by Dr. Patricia N. thing, Dr. Richard Schultz, art Dr. torrid Dean; see Mix A for papers contributed them authors. S ~ Pries of tats by Dr. John D. Biggers, Dr. Peter Braude, and Dr. Virginia Papaioannou; summary of talk by Dr. Harry M. Weitlauf, which was given ~ the fertilization section of the workshop, is combined ~ this chapter with talks included under implantation. Sac papers by Drs. Biggers and Papaioannou In Appendix A. For additional information, of Braude, P.R., Bolton, V.N., and Mbore, S., Human gene expression first coats between the fair- arxI eightball stages of pre implantation development. Nature 332:459-461, 1988; and Saute, P.R., Bolton, V.N., ark J*~n, M.H., the ~~-~ of human pry in infertility 1~. In Embryo Plead: Yes or No, Ciba Faction Sty of, G. BcxJk arX] M. Orator (FAR.) Tavis~c fess, Unit Kinsman, Ed. 63-82, 1986. 9. Chirp series of Calf given by Dr. Harry M. Wbitlauf, Dr. Jerome Strauss, arxl lDr. R. Michael Roberts. Sac pairs by Or. P0erts ark 1~5. ~tifaris, Strap-, jars} Klein In fix A. For additional detail ~ Weitlauf, H.M. and S~a~rt~n, M., Cafes In seated uterine proteins Fiats with embryo implantation in the ~se. Jaarnal of P=r~x~ti~ and Fertility. 84: 539-549, 1988. — 65 —