Nature Vol.

293 24 September 1981 253

Test-tube babies, 1981

R.G. Edwards*

Tu1s year should prove a turning point for patient, revealed by the different levels of stimulation can also distort the menstrual
the birth of children by the fertilization of sterioids in follicular fluids 7 and by vari- cycle, inducing a short luteal phase and a
human eggs in vitro. Between 15 and 20 ations in embryonic growth when super- disorganized endometrium, both incom-
such babies will be born in approximately ovulation techniques are applied to patible with establishing pregnancy 12 • An
equal numbers in the United Kingdom and animals. Some patients fail to respond to average of eight cycles of treatment with
Australia, and there will be one or two clomiphene. Others produce increasingly clomiphene is needed for oligomen-
elsewhere. These methods will be large amounts of oestrogens as several orrhoeic women to conceive naturally 3 •
introduced in many countries, primarily to follicles grow, and their endogenous LH Such disadvantages may be greater with
alleviate human infertility. Fundamental
aspects of human conception will be
Between fifteen and twenty babies will be born this year after the in vitro
analysed and increasing debate will
presumably be given to genetic fertilization of human eggs. Many of the essential steps now have high rates of
engineering. This is, therefore, an success, including the recovery of preovulatory oocytes, and fertilization and
appropriate time to assess the relevant embryo cleavage in vitro. Implantation of the embryo following its
clinical and scientific issues raised by this replacement in the mother remains the major difficulty. Some implications of
work 1 • the work are discussed.

The first essential: timing of surge stimulates ovulation. A difficult HMO than with clomiphene.
ovulation situation occurs in patients with moderate The alternative is to monitor the
I will first discuss the methods involved in or high levels of oestrogens and no approach of ovulation during the natural
timing ovulation for the collection of endogenous surge of LH (Fig. 1). As in menstrual cycle, and then aspirate the
preovulatory oocytes. other tissues 8- 11 , clomiphene may have single preovulatory oocyte. This method
Harvesting preovulatory oocytes is the depleted cytoplasmic oestrogen receptors was used for the conception of the first
first of several steps essential for obtaining in the pituitary gland of such patients over child by fertilization in vitro12 , and is widely
human embryos. They must be collected several days, so preventing the LH surge in used in our practice today. Plasma or
during their final stages of maturation just response to rising levels of oestrogens. urinary oestrogens are used to assess
before ovulation occurs, when meiosis is HCO must be given at some arbitrary time, follicle growth, and the onset of the LH
advanced and cortical granules have estab- before follicles become atretic, yet while it surge at mid-cycle provides a warning of
lished the defence against polyspermy. is uncertain if the patient will have her own the approach of ovulation.
Follicular growth and ovulation must be endogenous LH surge. Ovarian The disadvantages are obvious. There is

...-...... ....._. ..
regulated by endocrine therapy, or natural

. .. .. ., ..., ., ..
ovulation must be closely predicted during
the menstrual cycle.
The regulation of ovulation is
undoubtedly easier. Several follicles can be 400I
primed using human menopausal gonado-
tropin (HMO) or clomiphene early in the 300
menstrual cycle. An endogenous surge of
200
luteinizing hormone (LH) will then induce
ovulation. Alternatively, a single injection
100
of human chorionic gonadotropin
(HCO, 5,000 IU) can be given between
..
days 11 and 14, according to the follicular
response of each patient. Levels of urinary
b
.. 62~0 ~ :i: 7
.s... ...:i..c
. . •. . .... .., .. I4 -
oestrogens of 80-100 µg per day (refs ;:)
;:)
2,3), or follicular diameters of 1.5-2 cm
measured by ultrasound4-6 are believed to 0
be appropriate indications to inject HCO.
Ovulation can be induced at any desired
time of day or night, a considerable help in
organizing laboratory or surgical teams for 3 i::' ::--
oocyte recovery. Two or more pre-
ovulatory oocytes can be collected from
C • •-•• ••. - • ••• • •• •• .... ·•··· .... ..... ! ·2
•••
:,I J: I
1 ...J ..c::
0;:) 2
many patients, another advantage of
stimulating the ovary.
There may be problems to offset these
advantages. Wide variations exist in the Days
rate of growth of individual follicles in each
Fig. 1 Response of patients toclomiphene. a, Levels of urinary oestrogens rose considerably, and then fell in
the absence of an endogenous LH surge. b, Rising levels of urinary oestrogens followed by surge of LH in
*Physiological Laboratory, Cambridge University, Cambridge urine. c, No response in urinary oestrogens and no surge [' , Urinary oestrogens;•, urinary LH, measured
CB2 3EG. UK. and Bourn Hall, Cambridge CB3 7TR, UK. by the H i-Govanis kit.

© 1981 Nature Publishing Group

254 Nature Vol. 293 24 September 1981

follicular fluid helps to complete

Table 1 Aspiration of oocytes from preovulatory follicles
maturation, especially in those deliberately
Natural Natural and Clomiphene collected less than 26 h after the LH rise,
cycle• clomiphene cycle' which appear 'unripe'.
cycle'"
No. of patients 122
No. with accessible ovaries 109 Fertilization and cleavage
No. of preovulatory follicles aspirated 109 172 107 in vitro
No. (and 010) of oocytes collected 95 llO 96
(88"7o) (64"7o) (89"7o) Provided there are no pathological
conditions in the husband's spermatozoa,
*Recent series of 122 patients in Bourn Hall, Cambridge. Their average age was 33.9 yr. almost 90% of the preovulatory oocytes
aspirated during the natural cycle can be
fertilized in vitro (Table 2). The conditions
usually only one preovulatory oocyte, and lation, an obvious advantage in estab- of culture are rapidly becoming standard-
any pathological conditions in the ovary or lishing pregnancy. Several surveys have ized3·6· 7 • 12. Simple media are used,
abdomen will limit the chances of revealed a 1 in 4 chance of pregnancy including Earle's solution with pyruvate, a
collecting it. The LH surge is less predict- during unprotected intercourse in any medium designed to support mouse
able than an injection ofHCG, hence more menstrual cycle, but even this low rate may embryos in vitro 21 ·22 , and Ham's FlO, each
difficulty arises in organizing laboratory be higher than during induced cycles. supplemented with serum albumin or
and clinical staff to collect the oocyte. Clomiphene may be indicated in patients homologous human serum (7.5 - 8.60Jo).
Some difficulties have proved less than with irregular or prolonged cycles. We prefer Earle's solution containing
feared. Repeated blood sampling may pyruvate, with serum from each patient
provide a reliable guide for assaying Aspiration of oocytes (Table 3) 12 . Sperm numbers vary between
LH 6 •13 -15 , but is unnecessary, because urine A double aspirating needle or two separate 105 and 10' per ml, or between 1 and 5 x
needles are used, one channel being used 105 living spermatozoa per ml. Droplets of
Per cent for aspiration and the other to flush out the medium held under paraffin oil or small
50 follicle if the oocyte is not collected 19 ·20 • culture tubes can be used for fertilization.
The flushing solution may contain heparin, Our present practice is to begin fertilization
to prevent clotting within the follicle. in droplets of medium under oil, adding
The highest rates of collection are 1.5 - 2 x 105 living spermatozoa per ml
achieved 32 h after the injection of HCG, between 3 and 4 h after aspiration of
or 26 h after the rise of LH in urine (Table oocytes; the oocytes are placed in tubes
1). It is essential to time the onset of some hours later when most of their
ovulation correctly. Should HCG be given cumulus cells have been shed 12 .
after an endogenous surge of LH, The oocytes are fertilized within a few
ovulation may occur before aspiration hours. Some have been fertilized following
begins. Diurnal rhythms in tonic LH a re-insemination 48 h later and grew
2 release, sometimes reaching low surge normally to the eight-cell stage (J.M.
levels over a few hours, can confuse the Purdy and R.G.E., unpublished).
correct timing of the LH surge (Fig. 3). Conditions such as spermagglutination,
Pelvic adhesions, hydrosalpinx, cystic viscous seminal plasma and erratic sperm
follicles, endometriosis and other ovarian movement reduce the chances of
conditions impair the collection of oocytes, fertilization (Table 2). Almost all eggs are
and preliminary laparoscopy may be monospermic, indicating that the block to
required to alleviate these conditions. If the polyspermy is efficient3,6 , 23 .
ovary is accessible, oocytes can be collected Most embryos cleave normally. They
Time (h) during the natural cycle from almost 90% tolerate a wide variety of culture media,
of patients, laparoscopy being completed similar to those used for fertilization.
Fig.2 Diurnal rhythm in the onset of the urinary LH within a few minutes (Table 1). None of our Serum is almost universally added; in our
surge in women. The time shows the initial increase in patients had ovulated when laparoscopy work, we use 15% v/v of homologous
levels of l.H which was followed by a sustained rise (see
Figs lb and 3). was performed, and each of them had a serum 12 . Cleavage times are remarkably
large preovulatory follicle. similar despite the varying concentration
Preovulatory oocytes can be identified of energy sources in different media (Table
samples are suitable for rapid assays of LH quickly. They are embedded in a viscous 3). There have been occasional reports of
using the kit Hi-Gonavis (Mochida follicular fluid, to a diameter of 0.5 cm or cell fragments in cleaving eggs, although
Pharmaceuticals) 12 and for oestrogens. more and can be seen by eye2. The viscosity similar conditions have been found in
Fortunately, the LH surge begins in early of the cumulus mass serves as a guide to the embryos flushed from the reproductive
morning in almost three-quarters of the stage of maturity of the oocyte. A few tract and may not be pathological24 •25 .
women (Fig. 2), which is most convenient hours in culture in the presence of some Many embryos grow to morulae and
for the collection of preovulatory oocytes
24 - 28 h later. This 'critical period' in the Table 2 Fertilization of human eggs in vitro
LH surge in women resembles the situation
in rats 16 , 17 , and its distinct diurnal com- Patients with occluded oviducts Patients with
ponent must modify the feedback effects idiopathic
Bourn Hall* Melbourne' infertility'
of ovarian oestrogens and LH releasing
Total no. patients 95
hormone on the pituitary gland 18 • If No. with pathological conditions:
women discharge their LH at other times of Spermagglutination cells or debris in semen 5
day, their oocytes must sometimes be aspir- Abnormal movement of spermatozoa 3
ated less than 24 h later and cultured in Viscous seminal plasma I
Remainder 86 40
vitro to complete maturation. No. (and "lo) fertilized 76 (88"7o) 37 (92"7o) 350/o
Fertility may be higher during the
natural cycle than after ovarian stimu- *Recent survey of 122 patients (see Table I).

© 1981 Nature Publishing Group

Nature Vol. 293 24 September 1981 255
blastocysts, and escape from the zona
pellucid a in vitro 3 •6 ., although some
apparently develop abnormally during
these later stages of growth 6 • The fastest- 7
growing embryos are apparently the most
successful in establishing pregnancy6, 26 •
Cine films of cleavage with IR photo-
graphy may enable embryos to be exam-
ined for their nuclear structure before they
are replanted in the mother27 •

Replanting embryos
Implantation is the most difficult and
unpredictable stage. In animals, rates of 3
implantation are high when embryos are
replaced surgically (> 700Jo), but lower if
replaced non-surgically via the cervix
( - 500Jo )28 ,29 • Almost all human embryos
have been replaced transcervically, and
approximately one-quarter of them have I I

I I I I I I I I I I I I I I
Table 3 Composition of the media used for fertilization 8 88 8 88 8 88 8 88 8 88
and cleavage of human eggs in vitro (g ,- 1) g t-=N
o- g 0
t-=N
- 8o-
r......'.N 8 r-..: N
o- 8 r-: N
o-
Modified Media for Ham's
Time (h)
Earles 12 mouse FIO*
eggs21,22
CaCI22H2O 0.2649 0.441 Fig. 3 Diurnal variations in the levels of urinary LH in women. 'Tonic' levels rise each morning, then
KCI 0.400 0.356 0.285 decline. The LH surge began during the morning in this patient.
MgSO4.7H2O 0.200 0.294 0.1527
NaCl 6.800 5.140 7.400
NaH2PO4.2H2O 0.1583
Na2 HPO4 0.156 Physiological disorders might prevent Oldham33 , 7 in Australia6 •34 , and 2 recently
KH 2PO4 0.162 implantation. Vaginal distension during in the United Kingdom. Two of these were
NaHCO3 2.1 1.9 1.2 born prematurely at approximately 20
Ca lactate 0.527
replacement could invoke discharges of
Na pyruvate 0.011 0.025 0.110 prolactin, and a catheter passed through weeks, soon after amniocentesis. The
Glucose 1.000 1.000 1.100 the uterine cavity may invoke a premature children have been healthy, although a
Na lactate decidualization of the endometrium. Slight male twin required corrective surgery, and
(60"7o syrup) 3.7 ml 1- 1
myometrial contractions could expel several current pregnancies appear to be
Embryos growing most rapidly are two-cell by 21 h embryos soon after their replacement and progressing normally6 •26 • Details of all
after insemination, four-cell at 40 h, and eight-cell at might be inhibited using (J2 -mimetics30 , 31 • pregnancies are being recorded in an inter-
44-54 h (refs 3, 23). Restricting the volume of medium might national register to evaluate any risks to the
• Also contains CuSO4, FeSO4, ZnSO4 , amino acids, fetuses and the causes of abortion or fetal
etc.
encourage implantation (0.03 -0.10 ml are
used at present). Surgical transfer might death.
implanted in some series (Table 4). circumvent these problems, but a second Between one-quarter and two-fifths of
Embryos might be best replaced during the anaesthesia will be needed soon after that the fetuses arising from fertilization in
evening 12 , because the spontaneous con- used for oocyte recovery, and the myome- vitro die in utero, mostly in the first
tractility of the human uterus is lower at trium and endometrium will undoubtedly trimester. Maternal age (late thirties and
night 30 , 31 • Disorders in the luteal phase, bleed when a needle is passed through early forties) may be a disposing factor in
including progesterone deficiency and a them. In rhesus monkeys, 11 out of 15 some cases. One dead fetus was triploid 33 ,
short luteal phase, could preclude embryos replaced surgically in the oviduct, the others have not been karyotyped.
implantation (Table 4). Some implanted and 2 of 8 replaced in the uterus developed Triploidy may not be serious quanti-
embryos live for only a short time after the to advanced stages of gestation 32 • tatively, as the vast majority of fertilized
expected return of menstruation, and are eggs are monospermic. Most triploid
identified by a transitory rise in HCG{J (ref. Growth of human fetuses after fetuses arising during fertilization in vivo
3) (Table 4). replacement in the mother are caused by dispermy, fewer being due to
Why do so few embryos implant? At least 12 children have been born after fertilization by a diploid spermatozoon or
Perhaps 200Jo or thereabouts is all that can fertilization in vitro, 3 in the initial series in cleavage errors in the embryo 35 • Nor
be expected, since it is similar to the inci-
dence of natural human fertility reported
in serval surveys. This view seems to be Table 4 Pregnancies after replacing human embryos transcervically (same group of patients as in Tables 1 and 2)
pessimistic. Some embryos may be lost or
infected if there are difficulties in passing a Single catheter Double catheter
No. of patients 51 23
catheter through the internal os. An outer No. with deficient Juteal phases:
metal cannula can be useful in such cases, Short luteal phase• 2• 0
but pregnancy rates are lower (Table 4). A Progesterone deficiency t 1 3
Teflon catheter with a smooth tip and side No. with delayed [Link] menstruation+:
2 l (9.8"7o) 0
r,
Brief elevated levels of HCG/J
aperture evidently passes easily through the Brief elevated levels of HCG/LH 3 3
internal os6 , but may require the use of a No. pregnant 4 weeks after replacement 12 (23.l"lo) 2 (8.70/o)
tenaculum on the cervix. Implantations
have occurred after replacing all stages • Luteal phase of 12 days or less; these patients had luteal phases of 5 and 12 days, respectively, one also having
progesterone deficiency.
between the two-cell and blastocyst, but t These patients had between 0.4 and 2. 7 ng m1- 1 on days 7 or 8 of the luteal phase.
there are insufficient data as yet on the :j: Some other patients showed a delayed return to menstruation but no evidence was found of elevated HCG/J or
optimal stage for replacement 26 • HCG/LH.

© 1981 Nature Publishing Group

256 Nature Vol. 293 24 September 1981
The thought of cloned human embryos the genome of the fetus, but there is no
Table 5 Results on a recent series of 122 patients with
tubal occlusion
identical to a pre-existing individual is not information as to whether it was trans-
attractive. Yet, in a sense, uniparental cribed. There is apparently no knowledge
No. of patients 122 human embryos largely similar to the of the potential value of such treatments in
Failure to collect oocyte: father already exist. Hydatidiform moles preventing the expression of inherited
Method failure
Adhesions. endometriosis, etc.
14} 27
13
are the remains of diploid androgenetic conditions such as diabetes.
{Table I) fetuses, in which the trophoblast pro-
No. with preovulatory oocyt~s 95 liferates over several weeks to form large
Failure of fertilization
Method failure
grape-like vesicles. Such fetuses evidently Prospects
:+I?} 19 arise due to the expulsion of the female pro- Many children will soon be born after the
Pathological spermatozoa
(Table 2) nucleus from the fertilized egg43-46 • The fertilization of human eggs in vitro. We
Embryos not replaced 2 sperm chromosomes are doubled and the have established more than 40 pregnancies
No. of embryos replaced 74 embryo expresses paternal chromosomes since resuming work during the past 9
Failure of implantation:
only. Rare hydatidiform moles may even months, and the majority are surviving.
Short lutea! phase/
progesterone deficiency retain much of the father's heterozygosity, This is most encouraging for those couples
Method failure• 4:}
55 since they could arise from a diploid who could not be offered any other form of
Indications of pregnancy: spermatozoon. The reason androgenetic corrective surgery and have so far been
Delayed RTM: early abortion?
14
embryos undergo hydatidiform changes is without effective treatment (Table 5). The
Pregnant 4 weeks after replacement
not understood. Perhaps the embryonic method can be carried out several times on
The natural menstrual cycle was monitored. RTM, cells die early, or the mother 'rejects' a the same patient, and success rates should
return to menstruation. fetus lacking her own antigens46 ; never- soon exceed some forms of oviductal
•see Table 4. theless, androgenetic mouse embryos surgery. If ovarian stimulation is used,
develop normally to full term47 • Human 'spare embryos' may be available for
should trisomy be more frequent after gynogenones could also arise through embryological studies and one embryo has
fertilization in vitro. Most human trisomies similar processes involving expulsion of the grown for 9 days in vitro until stage 5a (ref.
arise during the first meiotic division of the male pronucleus from the egg, although no 3). The frozen storage of human embryos
oocyte 36 , 37 , a stage which is virtually searches have yet been made for them still appears to be distant. Some of the
complete before the oocytes are aspirated. before or after birth. Uniparental embryos fathers were oligospermic, and patients
Others arise during the second meiotic could arise through delayed syngamy and with idiopathic (unexplained) infertility,
divi'iion, that is, after fertilization, or an enlarged pronucleus, seen in two human hostile cervical mucus, incompetent cervix,
during cleavage of the embryos, but there is eggs fertilized in vitro. antibodies against the zona pellucida,
no record of their incidence in vitro. Subtle forms of genetic engineering have might also be helped. Complex disorders
Amniocentesis should be performed on all been introduced. DNA fragments con- leading to the abnormal growth of
fetuses arising through fertilization in taining the gene for human insulin were pronuclei can be investigated48 •
vitro, if the parents agree, until the risks of injected into pronucleate mouse eggs, and I thank Jean Purdy and Patrick Steptoe
trisomy have been assessed. the gene was identified in tissues of fetuses for their help at all stages of this work, and
Some fetuses may die in utero through at the 18th day of pregnancy42 • The DNA Simon Fishel for his comments on the
physiological or embryological factors evidently replicated and was inserted into manuscript.
associated with fertilization in vitro. These
could include damage to the uterine wall gynec. scand. 60, 109 (1981).
, I. 3rd World Congress of Human Reproduction, Berlin
during replacement of the embryos, the (Excerpta Medica, Amsterdam, 1981). 25. Wramsby, H. & Liedho!m, P. 3rd World Congress of
introduction of cervical flora into the 2. Edwards, R.G. & Steptoe, P.C. J. Reprod. Fert. Suppl. Human Reproduction, Berlin (Excerpta Medica,
22, 121 (!975); Lance/ i, 683 (1970). Amsterdam, 1981).
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embryo low in the uterine canaP. Short or (Academic, London, 1980). Congress of Human Reproduction, Berlin (1981).
4. Kratochwil, A., Urban, G. & Friedrich, F. Ann. Chir. 27. Hamberger, L. 3rd World Congress of Human Repro-
deficient luteal phases arising after ovarian duction, Berlin (Excerpta Medica, Amsterdam, 1981).
Gynaec. Fenniae61. 211 (1972).
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© 1981 Nature Publishing Group