Reproductive Technologies: I. Introduction
The development of effective and imaginative approaches to the management of human infertility has focused public attention on the techniques themselves and on their ethical and legal implications. Although differing widely in their complexity, these methods have one characteristic in common: the separation of human reproduction from the act of coitus. An understanding of these reproductive technologies is essential to an overall consideration of the ethical issues surrounding them.
Artificial insemination involves the mechanical placement of spermatozoa into the female reproductive tract. Inseminations are separated into two broad categories: those using the semen of the husband or designated partner (AIH) and those employing semen of a third party, or donor insemination (DI). Because the ethical and moral issues surrounding AIH and DI take on different dimensions, each will be considered separately.
AIH constitutes effective treatment when, for whatever reason, the male partner is unable to ejaculate within the vagina. Some males are unable to ejaculate during coitus but can ejaculate through masturbation or the use of vibratory stimuli. Certain anatomical abnormalities result in faulty semen placement. Hypospadias, a penile abnormality in which the opening of the urethra is located a distance from the tip of the glans penis, causes the ejaculate to be deposited at the periphery of the vagina even when the penis is well within. Retrograde ejaculation is a condition usually caused by a complication of prostatic surgery resulting in the formation of a channel that causes the ejaculate to be directed away from the penis and retrograded into the bladder. After ejaculation, semen for artificial insemination can be recovered from the bladder by catheterization.
Normal vaginal intercourse may be precluded by congenital or acquired vaginal abnormalities. In rare cases, the vagina is constricted as the result of in utero exposure to the hormone diethylstilbestrol (DES) or possibly by past trauma. Psychological problems in the male or female or both may interfere with normal coital exchange.
In recent years, AIH has been recommended when the semen displays deficiencies in numbers of sperm or their ability to move. Laboratory techniques have been developed to separate and concentrate the most active spermatozoa. These are then introduced into the uterine cavity, closer to the site of fertilization. Intrauterine insemination has been successful in cases of male infertility and in couples with unexplained infertility (Guzick et al.).
TECHNIQUES OF OBTAINING SEMEN. Semen for use in artificial insemination is usually obtained by masturbation. An alternate possibility is intercourse using a plastic condom. Coitus interruptus is not recommended, as the first portion of the ejaculate, which contains the majority of active, motile spermatozoa, is sometimes lost. In cases of obstruction of the vas deferens, which serves as the conduit for spermatozoa, spermatozoa can be obtained surgically from the epididymis, the storage depot for spermatozoa. Specimens so retrieved have been used successfully for in vitro fertilization.
TIMING OF THE INSEMINATION. Placement of spermatozoa should be timed to coincide with the twelve hours immediately preceding ovulation. Approximately twenty-four hours before ovulation, increased levels of luteinizing hormone can be detected in the urine, using a color indicator to predict ovulation. The day-to-day development of the egg-containing ovarian follicle can be monitored with pelvic ultrasound. To enhance the accuracy of ovulation timing still further while causing the release of additional eggs for fertilization, the use of human gonadotropins to induce ovulation has become increasingly popular.
INSEMINATION AND SEX SELECTION. Insemination has also been used with limited success for sex selection. Laboratory methods have been suggested to separate the X-chromosome-bearing (female-producing) from the Y-chromosome-bearing (male-producing) spermatozoa. Success rates in the production of male offspring in the 80 percent range are claimed (van Kooij and van Oost). Such techniques are useful in animal husbandry but do not yield a consistently satisfactory success rate in humans. Sex selection would be useful to avoid a sex-linked genetic disease. Sex preselection based solely on preference for a boy or a girl has much wider social implications.
DONOR INSEMINATION. Donor insemination was mentioned as a method of treating infertility in the nineteenth century. As DI has become more widely used, the legal climate has become more favorable and the status of the offspring much less uncertain. With this has come awareness of the importance of careful counseling and the use of appropriate permission forms. There has not yet been a case in U.S. law in which the anonymous sperm donor has been assigned parental responsibility.
The clinical indications for donor insemination are related mainly to deficiencies in the semen. The most clear-cut cases are those in which the male partner suffers from azoospermia (absence of spermatozoa). Indications have been extended to include those in whom some spermatozoa are present but the quality of the specimen is poor. Known hereditary disorders in the male partner, such as Huntington's disease, Tay-Sachs disease, or hemophilia, are also indications for DI.
In vitro fertilization (IVF) has widened the possibility of conception with severely deficient semen. Donor insemination is sometimes used in IVF when there is failure of fertilization using the male partner's specimen.
EVALUATION OF THE COUPLE FOR DONOR INSEMINATION. A couple considering donor insemination should be thoroughly counseled. If either partner has reservations, it is wise to accept these at face value and encourage consideration of other options, including adoption. The man's fertility should be thoroughly evaluated, and efforts made to correct any abnormalities. The woman also should be thoroughly evaluated for factors that might contribute to infertility. Both partners are usually required to review and sign a detailed informed-consent form.
SELECTION AND SCREENING OF DONORS. Unless he expresses willingness to be identified, the donor is anonymous. Occasionally there is a request that a close relative (usually a brother or even a father) be used. In such cases, the couple should be encouraged to consider carefully the potential for future familial conflicts. Analysis of donor semen should meet the normal standards for fertility (ASRM, 2002). The donor should be in excellent health and be screened for any family history of genetic disorders. Serologic tests for syphilis and serum hepatitis B antigen are obtained initially and after six months. The genitalia are cultured for gonorrhea and chlamydia. An initial screening for the AIDS virus antibodies is performed and repeated after six months because the antibody test for AIDS may not turn positive until several months after infection. Most centers now use frozen semen exclusively. If a donor is providing repeated specimens, periodic reevaluation of his health status is essential. Clinics should maintain records of pregnancies and set a limit on the number of pregnancies any one donor may produce. To decrease the possibility of consanguinity (procreation between close relatives, such as siblings or first cousins) in a given population, an arbitrary limit of ten or fewer pregnancies is recommended.
It is important to maintain confidential donor records, including all of the information on the screening procedures, so that it is available in the future in case it is needed for medical reasons.
TECHNIQUE OF INSEMINATION. The standard insemination involves placing the specimen, thawed if it has been frozen, into the cervical canal by means of a small, flexible tube (cannula). As the vaginal speculum is removed, the remainder of the specimen is placed in the vagina, at the outer cervical canal. The patient remains supine for twenty minutes or so. The specimen may be held in place with a cervical cap, which is removed four to six hours after insemination. For intrauterine insemination, a plastic cannula is passed through the opening of the cervix into the uterine cavity, where the concentrated, pretreated (i.e., washed) spermatozoa are deposited.
CRYOPRESERVATION OF SEMEN. Since the first successful insemination with freeze-stored semen in 1953, this technique has had a significant impact on clinical practice. In the 1970s, formal semen banks were established, largely to address the needs for long-term preservation of the specimens of men who had undergone vasectomy. Semen also is preserved prior to chemotherapy or radiation, which might result in sterility. Although there is no formal reporting system, information accumulated over the years has failed to uncover an increased incidence of genetic defects among the offspring resulting from insemination with cryopreserved semen.
The response of spermatozoa to cryopreservation is unpredictable and varies on an individual basis. Some specimens freeze well and others do not. The pregnancy rate is lower overall with frozen semen. The only reliable way to determine whether a specimen is suitable for cryopreservation is to cryopreserve it, thaw it, and evaluate the impact of the procedure on the quality of sperm motility. Specimens are usually stored in individual straws or small vials so that fractions may be thawed while the remainder is preserved for future use. The Ethics Committee of the American Society for Reproductive Medicine (formerly the American Fertility Society, AFS) has determined that cryopreservation of human semen is ethically and medically acceptable (ASRM, 2002). Most programs use only cryopreserved semen for donor insemination.
In Vitro Fertilization
In vitro fertilization and embryo transfer (IVF-ET) is increasingly common in infertility practice. Initially used exclusively in women with damaged fallopian tubes, the indications for IVF-ET have been extended to include male factor infertility and cases in which no cause for the infertility can be uncovered. Much as artificial insemination separates procreation from the coital act, in vitro fertilization separates fertilization from the normal maternal environment, allowing the initial phases of development to occur outside the reproductive tract, followed by transfer of the embryo into the uterus. The first successful in vitro fertilization was carried out in a normally ovulating woman whose tubes had been surgically removed. A single egg (ovum, oocyte) was obtained by aspiration at the time of laparoscopy. The procedure required general anesthesia and involved placing a telescope through the umbilicus for visualization of the pelvic structures. The oocyte was fertilized in vitro and transferred to the uterus after two days.
In later developments, the ovaries were stimulated with human urinary gonadotropins to induce development of several follicles, each containing an ovum, in a given cycle. This approach is now standard. Follicular development is followed by means of blood estrogen levels, and the size of the growing follicles is measured by ultrasound. When the follicles are judged ready for ovulation, a second hormone, human chorionic gonadotropin, is administered to induce ovulation. This causes further development of the follicles and the maturing of oocytes within them. The oocytes complete their first division in a process referred to as meiosis, releasing half their complement of chromosomes in a small, round structure, the first polar body. The maternal chromosomes are now ready for the second meiotic division, which occurs after the ovum has been penetrated by the spermatozoa. Within two to three hours of the expected time of ovulation, the oocytes are aspirated from their follicles.
In the early phases of IVF development, this was carried out with the aid of the laparoscope. The oocytes were obtained by needle aspiration. Today, ova are obtained by ultrasound-guided transvaginal aspiration. This procedure can be done without general anesthesia, and the overall approach to in vitro fertilization is greatly simplified.
Another major clinical problem in the early phases of IVF development was that occasionally a patient would ovulate before the oocytes could be obtained, and the cycle would have to be canceled. Analogues of the gonadotropinreleasing hormone are now used to prevent this. These analogues are capable of blocking the release of the patient's pituitary gonadotropins, and the ovaries can be brought under the complete control of exogenously administered hormones. The number of follicles that develop varies from patient to patient, and even in the same patient from one cycle to the next. By and large, the aim is to obtain as many oocytes as possible in a given treatment cycle, especially if the couple has selected cryopreservation as a possible option.
IVF treatment is both physically and emotionally demanding. Several visits for hormone determinations and ultrasound are required. Ovum recovery, although relatively safe, is not without complications. Rarely ovarian infection occurs, which can further compromise the fertility status of the patient. This point is particularly pertinent when oocytes are being obtained for donation.
A freshly ejaculated semen specimen is obtained for insemination. The ova are placed in individual containers and mixed with spermatozoa that have been prepared by separating them from the semen and incubating them in a solution designed to enhance their fertilizability. The inseminated ova are cultured for approximately twenty-four hours and then inspected for evidence of fertilization.
Much has been learned about human fertilization through in vitro fertilization. When it is removed from the woman's body, the ovum is surrounded by layers of small, loosely packed cells, the cumulus oophorus. An inner layer of more densely arranged cells, the corona radiata, immediately surrounds the oocyte. These cells interface with the zona pellucida, a translucent protein shell that immediately surrounds the egg. Penetration past these barriers is accomplished through a sequence of interactions between spermatozoa and the ovum and its layers (Kopf and Gerton). When the spermatozoon reaches the zona pellucida, a series of chemical communications occurs. These condition the spermatozoon so that it can penetrate through the zona pellucida. Once past the zona, the spermatozoon attaches to the egg membrane and is then incorporated into the egg cytoplasm, the tail along with the head. The head is then transformed into a pronucleus. The second polar body is released and the nucleus of the egg is transformed into a pronucleus. The pronuclei then join and the chromosomes are intermingled in preparation for the first cell division. Twenty-four hours after insemination, there are two pronuclei and two polar bodies. This constitutes evidence that the penetration has been successful and fertilization is in process. After three days, the embryo has developed to the eight-to sixteen-cell stage and is ready for transfer into the uterus. Transfer is sometimes delayed until day five or six to allow growth to the blastocyst stage.
EMBRYO TRANSFER. The dividing embryos are incorporated into the end of a catheter that is then passed through the cervical opening into the uterine cavity, where they are discharged. The pregnancy rate is progressively improved if more than one embryo is transferred. If more than three are transferred, there is a greatly increased possibility of multiple pregnancy. Twins are not usually a problem, but triplets or more greatly increase the possibility of fetal loss. Therefore, in many IVF programs no more than two fertilized oocytes are transferred in women under age thirty-five and three in the older group. The availability of cryopreservation has made such decisions easier.
Moral Status of the Embryo
The issue of when meaningful human life begins is pivotal in any discussion of IVF. The fertilization process is a complex series of events. The spermatozoon must be exposed to the environment of the female reproductive tract for a period of time before it acquires the ability to penetrate the layers surrounding the recently ovulated oocyte. This process, referred to as capacitation, takes between one and two hours in the human. It is reproduced in vitro in the fluids used for sperm preparation. The series of events involving penetration through the zona pellucida requires complex chemical communication between sperm and egg. After the spermatozoon has penetrated into the cytoplasm, completion of fertilization, although increasingly probable, is not assured.
The events that follow, including the formation and subsequent fusion of the pronuclei, occupy more than twenty-four hours. In the natural sequence of events, the conceptus remains in the fallopian tube for approximately three days. At the eight-to-sixteen-cell stage, it is transported into the uterus. There it develops into a fluid-filled structure, the blastocyst, that attaches to the uterine lining, or endometrium, on the sixth to seventh day after fertilization. The blastocyst is incorporated into the endometrium and invades blood vessels. Development occurs rapidly thereafter, but it is not until the fourteenth day that it develops unique characteristics. This coincides with the formation of the primitive streak, a linear region that can be identified on the early embryonic disk; it signals the beginning of the development of a distinct category of cells. Until this point, there is the potential for division into identical twins. Each of the individual cells in the early conceptus has the potential to develop into a complete adult. On or about day five or six, specialized cells, the trophoblasts, are formed. They provide the point of attachment for the placenta and are essential to the nourishment of the growing embryo. The Ethics Committee of the American Society for Reproductive Medicine applies the term pre-embryo to the conceptus through the first two weeks of gestation (AFS). It takes the position that the moral status of the pre-embryo is different from that of either the unfertilized eggs and spermatozoa or the later stages in embryonic development.
Cryopreservation of Pre-embryos
Techniques for freeze-preserving pre-embryos have contributed to the success of human in vitro fertilization and embryo transfer. The incidence of multiple pregnancy, which increases dramatically if more than two to three preembryos are transferred, can be reduced with the availability of cryopreservation. Pre-embryos not transferred during the treatment cycle can be used in subsequent spontaneous ovulation cycles. When pregnancy occurs in the initial treatment cycle and pre-embryos have been cryopreserved, a number of future options must be considered. These issues should be reviewed and decisions made before the preembryos are frozen. Patients whose response to stimulation clearly indicates that more than three oocytes will be recovered should consider the freezing option well in advance of ovum recovery. Those who for whatever reason, including deeply felt moral reservations, choose not to cryopreserve may wish to have sperm added to no more than three oocytes and have all of the fertilized specimens transferred. Remaining ova can be disposed of in their unfertilized state. Another alternative short of cryopreservation is to fertilize all available ova and select only the best of the resulting preembryos, as determined by their appearance and rate of cell division, for replacement, discarding the remainder.
The standard consent form should contain a detailed description of the possibilities to consider if a decision is made to cryopreserve human pre-embryos. As far as is known, cryopreservation of human pre-embryos is not associated with adverse fetal effects. Generally it is agreed that the pre-embryos will be frozen and stored for use in subsequent cycles. Unforeseen situations can occur, such as failure of equipment, although backup freezer systems and liquid-nitrogen holding facilities are usually available in the event of such an occurrence.
In most major centers, the disposition of unused frozen pre-embryos is reviewed in advance of cryopreservation. Handling of these pre-embryos is subject to the couple's joint disposition. They agree that if one partner is unwilling or unable to assume responsibility for the fertilized eggs, the responsibility reverts to the other partner. If that person is not willing or able to assume ownership, the hospital or clinic usually reserves the right to dispose of the pre-embryos in accordance with policies in existence at the time.
Micromanipulation of Oocytes and Embryos In Vitro
Instruments have been developed to allow manipulation of gametes and pre-embryos under magnification. These techniques of micromanipulation have been used extensively in laboratory mammals. More recently they have been applied to human eggs, spermatozoa, and pre-embryos. When the oocyte is not penetrated by spermatozoa that are otherwise apparently normal, micromanipulation can be used to insert a spermatozoon mechanically through the zona pellucida directly into the oocyte itself, a technique known as intracytoplasmic sperm insertion (ICSI). In males with a congenitally obstructed vas deferens, sperm may be recovered directly from the epididymis and used for ICSI. Pregnancies that would otherwise be impossible can occur as a result of these procedures. Because abnormalities in the semen and vas obstruction may be associated with genetic risk factors, these should be considered before proceeding with ICSI (Dohle et al.).
Micromanipulation has been extended to pre-embryos. It has been suggested that the second polar body, the cell that is released from the ovum at the time it is penetrated by the spermatozoon, be removed for chromosome analysis in an effort to determine whether the embryo is genetically normal. This approach could be used in couples at risk of genetic abnormalities and would avoid the onus of a decision to terminate the pregnancy later on. Individual cells have been removed from the embryo for analysis without apparent harm (Tarin and Handyside). Other possibilities may eventually emerge, including the removal and storage of individual cells as clones of the embryo that is transferred. Some of these approaches have not yet attained clinical practicality, but they raise moral, ethical, and legal issues that it would be wise to address now.
Gamete Intrafallopian Tube Transfer
The procedure referred to as gamete intrafallopian tube transfer (GIFT) involves the transfer of freshly recovered ova and conditioned spermatozoa into the fallopian tubes. Thus, fertilization actually occurs in vivo. GIFT is not applicable to all infertility patients. Those with damaged or absent fallopian tubes are obviously not candidates. GIFT has been recommended for couples with unexplained infertility and women with extratubal disease, such as pelvic adhesions or endometriosis. Although fertilization occurs within the fallopian tube, GIFT is certainly assisted reproductive technology and is clearly separated from the coital act. When more than four ova are recovered at the time of a GIFT procedure, one or more are usually fertilized in vitro and cryopreserved for transfer in subsequent cycles. Transfer of the ova and spermatozoa into the fallopian tubes is usually carried out by means of laparoscopy. The success rate following GIFT is now surpassed by that of in vitro fertilization (SART/ASRM). In most centers, GIFT is now largely supplanted by IVF.
Surrogate Gestational Mothers
Human in vitro fertilization has opened the possibility that the resulting pre-embryos can be transferred to a woman other than the woman providing the oocytes. The second woman, referred to variously as a surrogate carrier, a womb mother, a placental mother, or a surrogate gestational mother, provides the gestational but not the genetic component of that pregnancy. Usually arrangements are made for the couple whose egg and sperm produced the embryo to adopt the newborn.
In another type of surrogacy, a husband's spermatozoa are used to inseminate a woman other than his wife. This surrogate mother carries the gestation to term. Agreement is reached before the procedure is carried out that the contracting couple will have custody of the resulting child.
In everyday infertility practice, there are circumstances that seem to justify these procedures. Consider a woman who was born without a uterus but with normal, functioning ovaries. Her husband is normally fertile. The patient's sister had a tubal sterilization after three pregnancies and is healthy in every way. The patient's sister's husband is entirely in agreement with the patient's sister's desire to act as a gestational surrogate mother. Oocytes are obtained from the patient, they are fertilized with her husband's spermatozoa, and the pre-embryos are transferred to her sister's uterus. In this situation we are virtually 100 percent confident that the pregnancy resulted from the procedure and is not an accidental result of coitus between the surrogate and her husband. The offspring is the genetic product of the husband and wife and has no direct genetic relationship to the patient's sister.
Other cases involve the use of a surrogate mother who contributes 50 percent of the chromosomal makeup of the offspring; this represents a more complex situation. The birth mother, who clearly is genetically related to the offspring, will be giving up her newborn child (hers in terms of both birth process and genetics). Indications for the use of a surrogate gestational mother include any condition in which there are functioning ovaries but an absent or nonfunctioning uterus. The uterus may be congenitally absent or may have been removed because of disease; it may be nonfunctional as a result of in utero DES exposure. A surrogate carrier may also be considered if pregnancy is illadvised for reasons of maternal health. Another issue concerns responsibility for the child in the event that the child is abnormal or damaged as a result of premature birth or birth trauma. There are also issues of the health status and behavior of the surrogate gestational mother during pregnancy. One must consider the impact of drugs or alcohol and the possibility of transmission of diseases. Finally, there is the matter of payment to the surrogate gestational mother. The possibility for exploitation certainly exists.
The clinical indications for the use of donor ova usually are rather straightforward. They include premature menopause and the inability of the wife to produce genetically normal oocytes. On the surface, the ethical issues surrounding the use of donor oocytes should be no different from those involved in the use of donor semen. They are compounded, however, by the risks involved in obtaining oocytes compared with obtaining a semen specimen. For example, ovarian infection could occur following ovum retrieval, which could result in permanent sterility (Tureck et al.). In addition to the cost of the procedures, which is usually borne by the couple requiring the oocytes, there is also the question of payment to the donor for her time, pain, and suffering.
In contrast to spermatozoa, oocytes are difficult to cryopreserve; hence, menstrual cycle coordination between the recipient and the donor is required. Alternatively, donor oocytes may be fertilized with the husband's sperm, and the pre-embryos cryopreserved for future transfer. Sources of donor oocytes include the excess eggs from patients undergoing IVF, oocytes obtained incidental to an operative procedure such as a sterilization, or a specific donation by a relative or close friend. Increasingly, the source of the eggs is a paid "volunteer" (ASRM Ethics Committee). The availability of this technology allows pregnancy in women who are well past the ordinary childbearing age (Sauer, Paulson, and Lobo).
In an effort to improve oocyte quality, cytoplasmic transfer between human oocytes, that is, ooplasm donation, has been attempted. The procedure involves aspirating cytoplasm, the portion of the egg surrounding but not including the nucleus, from a donor egg and injecting it into a recipient egg. Recipient oocytes were deemed to be of poor quality or were recovered from women in their late reproductive years or who previously had a failed IVF cycle. Not unlike some of the early approaches to IVF, the procedure was carried out with minimal basic research background, although in limited studies the technique was found not to impair successful fertilization and cell division in the mouse. Unfortunately, children born as a result of this technique have now exhibited traces of mitochondrial DNA from the donor egg. This foreign cytoplasmic DNA may result in untoward consequences in the future and, defects that are transmitted might be heritable and therefore could be observed in the next generation. Until and unless the safety and efficiency of this approach is established in suitable animal models, this effort to rejuvenate deficient oocytes must be approached with extreme caution.
The techniques employed in what is known as the new assisted reproductive technologies are varied and challenging. They range in complexity from seemingly straightforward artificial insemination to micromanipulation of ova, spermatozoa, and pre-embryos—and perhaps, in the future, to treatment of genetic disease by gene insertion in vitro. Just as the techniques vary, so do the ethical issues surrounding them. In no other field is there a greater opportunity for interaction among the physician-scientist, ethicist, moral theologian, social scientist, and legal scholar.
luigi mastroianni, jr. (1995)
revised by author
SEE ALSO: Abortion; Adoption; Christianity, Bioethics in; Cloning; Embryo and Fetus; Fetal Research; Genetic Testing and Screening: Reproductive Genetic Testing; Maternal-Fetal Relationship; Moral Status; Transhumanism and Posthumanism;Women, Contemporary Issues of; and other Reproductive Technologies subentries
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American Society for Reproductive Medicine (ASRM). Ethics Committee. 2000. "Financial Incentives in Recruitment of Oocyte Donors." Fertility and Sterility 74(2): 216–220.
Cohen, J.; Scott, R.; Schimmel, T.; et al. 1997. "Birth of Infant after Transfer of Anucleate Donor Oocyte Cytoplasm into Recipient Eggs." Lancet 350: 186–187.
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Guzick, D. S.; Carson, S. A.; Coutifaris, C.; et al. 1999. "Efficacy of Superovulation and Intrauterine Insemination in the Treatment of Infertility." New England Journal of Medicine 340: 177–183.
Hawes, S. M.; Sapienza, C.; and Latham, K. E. 2002. "Ooplasmic Donation in Humans: The Potential for Epigenic Modifications." Human Reproduction 17: 850–852.
Kopf, Gregory S., and Gerton, George L. 1990. "The Mammalian Sperm Acrosome and the Acrosome Reaction." In The Biology and Chemistry of Mammalian Fertilization, ed. Paul M. Wasserman. Boca Raton, FL: CRC Press.
Sauer, Mark V.; Paulson, Richard J.; and Lobo, Rogerio A. 1990. "A Preliminary Report on Oocyte Donation Extending Reproductive Potential to Women over 40." New England Journal of Medicine 323(17): 1157–1160.
Sauer, Mark V.; Paulson, Richard J.; and Lobo; Rogerio A. 1993. "Pregnancy after Age 50: Application of Oocyte Donation to Women after Natural Menopause." Lancet 341(8841): 321–323.
Society for Assisted Reproductive Technology (SART) and the American Society for Reproductive Medicine (ASRM). 2002. "Assisted Reproductive Technology in the United States and Canada: 1998 Results from the American Society for Reproductive Medicine/Society for Assisted Reproductive Technology Registry." Fertility and Sterility 77(1): 18–30.
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