2. Susceptible periods in women

SUSCEPTIBILITY TO MUTATION AROUND OVULATION

There is a period immediately around ovulation when the maturing ovum is particularly easily damaged by many kinds of environmental hazard. Work sponsored by the US Atomic Energy Commission at the Oak Ridge National Laboratory showed that mice were highly sensitive to radiation during the 8.5 hours before mating which coincides with ovulation. Liane Russell (1956) wrote:

"Females irradiated 8.5 hours before fertilization produced only about one sixtieth as many living embryos as those irradiated at any stage 16 hours to 4bd days before fertilization."

In another paper the Russells wrote that a 70-fold increase in sensitivity occurs between 11.30 a.m. and 7 p.m. on the day preceding ovulation (Russell & Russell, 1956). These studies showed that the deaths of mice ova caused by radiation before ovulation were associated with damage to chromosomes. The results of animal experiments provide warnings that need when possible to be confirmed, but warnings that are to be taken seriously until shown to be wrong. The more species of animal to which some generalization applies the stronger is the hypothesis it generates for humans. The increase in susceptibility to environmental hazards during preovulatory maturation is shared by all mammals that have so far been the subject of experiment including other primates, but with differences between species in the length of the period of susceptibility.

The Russells' experiments were only concerned with the effects of radiation. A colleague of the Russells found that the susceptibility to chemicals also increased during the period before mating and around ovulation, and Figure 2.1 is based on his research (Generoso, 1969). Female mice were divided into 4 groups and were each given a dose of a mutagen at different times before ovulation. In the fourth group, dosed with the mutagenic chemical between 12 hours and 5 days before ovulation, it is seen that over 80 per cent of ova suffered mutations which were lethal, that is the embryo resulting from fertilization was dead or unable to survive for long. In contrast, it is seen that only 10 per cent had lethal mutations if the female mice were dosed with the mutagens from 15 to 20 days before ovulation. Fig 2.1 suggests that the shorter the interval between exposure to some hazard and ovulation, the greater the risk to the outcome of pregnancy. When thinking of humans, the timing is different from animals, but the principle is the same.

Chemicals, unlike radiation, produce a somewhat blurred picture of the period of susceptibility around ovulation because they take time to reach and affect the ovum and usually disappear quite slowly over hours or days. Pulses of radiation show the quite rapid changes in susceptibility more clearly. The beginning of preovulatory maturation is marked by a rapid and substantial rise in the release of two pituitary hormones, follicle stimulating hormone (FSH) and luteinizing hormone (LH). The peak of LH release is shown in Figure 2.2. using data showing the radio-sensitivity of the ova of Chinese hamsters from a study by Mikamo (1982) of Asahikawa Medical College.

A pulse of radiation was given at 15 different times to separate groups of animals during the 84 hours before ovulation. The percentage of chromosomal abnormalities was recorded in ova recovered about 5 hours following ovulation at metaphase II without mating. Only animals maintaining a regular oestrous cycle were used. The peak of chromosomal aberrations caused by radiation is seen to coincide with exposure to a dose of radiation around the peak of the LH surge, about 10 hours before ovulation. During these 10 hours Mikamo's hamsters showed an increase in susceptibility to radiation of about 60 times. This is the same increase in susceptibility that the Russells reported in 1956. The time from the LH surge until ovulation includes meiosis I and the early preparatory stages of meiosis II. Not only mammals but some invertebrates have been shown to share this increased susceptibility to insult during the two stages of meiosis. In the male meiosis is also a time of high susceptibility to environmental hazards.

In Figure 2.2 the effect of radiation on the ova of non-mated female hamsters is shown. In later experiments reported in the same paper Mikamo and his team studied the numbers of embryos surviving 18.5. days, instead of 5 hours, after ovulation and mating, using the same doses of radiation at the same times before ovulation. Mikamo found the same period of high susceptibility in mated animals coinciding with ovulatory maturation, but found more deaths of embryos or "dominant lethal mutations" than they expected from the chromosomal abnormalities measured in the previous experiments, particularly for the doses of radiation during the 8 hours before ovulation. Chromosomal abnormalities are generally lethal. It appeared that some of the effects of radiation took more than a few hours to become apparent. These further experiments emphasized that the high susceptibility persists through the whole period from LH surge until meiosis II.

SUSCEPTIBILITY TO MUTATION AROUND CONCEPTION

Meiosis II is completed in humans after a sperm enters the ovum. The chromosomal abnormalities reported in these expenments were "new mutations" caused by radiation, following the definitions in Chapter One. The story is taken a step further in Figure 2.3 based on another study by Generoso which begins where Mikamo left off (Generoso et al., 1987). The high susceptibility to poisons in mice is seen in Figure 2.3 to continue after mating. Generoso commented that it was at one time generally believed that chemicals and radiation only produce congenital abnormalities in surviving embryos much later after implantation of the ovum in the uterus when the embryo is actually producing the abnormal organs and limbs. "This long-standing belief" said Generoso, was obviously wrong because he and his colleagues had succeeded in producing "remarkable increases in the incidence of congenital abnormalities

and death of foetuses" by exposure to a mutagen only 1 hour or 6 hours after mating and not later in the embryonic period. At the time when Generoso exposed his mice to chemicals the fertilized ova were still only single cells. The organs and limbs did not yet exist except in the genetic code. Generoso suggests that damage by mutagens at this time is genetic damage, or in other words new mutations, causing the subsequent foetal loss. Mutations continue indeed to happen for a time after fertilization. Generoso in the experiments illustrated in Figure 2.3 used ethylene oxide as the mutagen. Ethylene oxide is an important industrial chemical used in the production of ethylene glycol, antifreeze for motor vehicles, and acrylic ester fibres. It is also used as a fumigant for foodstuffs, textiles and in the sterilization of surgical instruments. The interval between mating Generoso's mice and sperm entering ova is shown as only one hour. The mice were in 4 groups, the first given ethylene oxide an hour after mating the fourth around first cleavage. In women the interval may be 24 hours or even longer, and the time to first cleavage when the fertilized ovum divides for the first time may be 2 or 3 days. In Generoso's experiments exposure I or 6 hours after fertilization greatly increased the number of late foetal deaths as shown in Figure 2.3, but losses were much lower for exposure beginning 9 hours after fertilization and were not different from controls for exposure beginning 25 hours afterwards. These studies were repeated by the same team using three other chemical mutagens all of which produced developmental abnormalities following exposure of early zygotes. Two of the mutagens also produced high losses of conceptuses in all postmating stages up to implantation and later (Generoso et al., 1988; Katoh et al., 1989). A dilute solution of alcohol fed to female mice during this susceptible period around and immediately after mating has been shown by Kaufman in Cambridge (1983) to cause chromosomal abnormalities.

How long then, is the period of highest susceptibility in women? Figure 2.4. is an attempt to put the evidence together in a simplified picture of the susceptibility of women around ovulation. A safety factor is added at each end of the period. The time from ovulation to first cleavage, that is to completion of the first cell division to produce a 2-cell embryo, is normally about 1bd days (McLaren, 1982). This time can, however, be longer, particularly because the time between ovulation and fertilization may be longer. The time from ovulation to first cleavage is therefore shown conservatively as 2bd days. In women the time between the peak of the LH surge and ovulation is reported to be 30 to 36 hours (Edwards & Steptoe, 1975). In the interests of caution 36 hours is used in Figure 2.4. Assuming the human time-scale prior to ovulation is 3.3 times longer than that of the Chinese hamster the period of highest susceptibility of women would be as long as 60 hours prior to ovulation. The beginning of the period of highest susceptibility in Figure 2.4 is shown as 3 days, counting backwards from ovulation and allowing for some variation in the speed of events. Counting forwards from ovulation it is seen that the susceptibility is already decreasing before the first cleavage. It is therefore suggested that the whole period of highest susceptibility in Figure 2.4 lasts about 4bd days. The time of ovulation is usually about mid-cycle, or about 14 days after the first day of menses in a 28-day cycle. The length of cycle varies from under 25 days to over 35 days and the post-ovulatory part of the cycle varies between about 10 and 14 days.

A report on the animal testing of chemicals for mutagenicity proposed that initial testing should be restricted to the period around meiosis I and postmeiotic stages as no chemical has ever been shown to induce mutations which fails to do so during these active stages of meiosis (Epstein & R6hrbom, 1970). Furthermore chemicals that induce mutations during these mejotic stages may not produce any evidence of mutagenicity at other times at comparable doses. This report was, however, only concerned with dominant lethal testing and chromosomal abnormalities and to the exposure of mainly male animals to chemical mutagens. This kind of testing does not identify chemicals producing only gene mutations or deletions and can therefore only provide positive evidence of mutagenicity and can never show that a chemical is not mutagenic.

Mikamo counted lethal mutations but did not study mutations in survivors. Nomura (1982) of the Institute for Cancer Research, Osaka University, used both X-rays and a chemical mutagen, urethane, on both male and female mice before mating and studied congenital malformations and tumours in survivors. Urethane is a potent chemical mutagen but nevertheless does not cause dominant lethal mutations or chromosomal abnormalities. Examination of 5,830 embryos by Nomura revealed no significant increase of dominant lethal mutations following dosing of a parent with urethane. X-rays on the contrary produced dominant lethal mutations as expected. Most malformations and tumours are, however, caused by gene mutations not by visible chromosomal abnormalities and urethane given to male or female mice before mating was very effective in producing malformations as shown in Figure 2.5 and tumours as shown in Figure 2.6. In one series of Nomura's experiments malformations were recorded in 19-day old foetuses, as shown in Figure 2.5., and in a second series in 7-day old offspring.

The rate of malformations fell by 50 per cent between 19 day old foetuses and 7 day old pups because of the high mortality shortly after birth. At 7 days there were more than 10 times as many malformed pups as in controls. One of the less lethal malformations, open eyelid, was shown to be heritable. The tumours were diagnosed in offspring at 8 months of age and were found to be heritable in experiments confined to the male line as far as the F3 generation with a dominant pattern of inheritance and about 40 per cent penetrance. The strain of animals used may have had a particular susceptibility to tumour initiation. It is apparent, however, from these experiments that tumours in offspring can be produced by exposure of the parental germ line to mutagens before fertilization. The females were irradiated or dosed with urethane in what was described as the "late follicular stage" which covered the 14 days before ovulation. As there are profound changes in female susceptibility during these 14 days Nomura varies from under 25 days to over 35 days and the post-ovulatory part of the cycle varies between about 10 and 14 days.

A report on the animal testing of chemicals for mutagenicity proposed that initial testing should be restricted to the period around meiosis I and postmeiotic stages as no chemical has ever been shown to induce mutations which fails to do so during these active stages of meiosis (Epstein & R6hrbom, 1970). Furthermore chemicals that induce mutations during these mejotic stages may not produce any evidence of mutagenicity at other times at comparable doses. This report was, however, only concerned with dominant lethal testing and chromosomal abnormalities and to the exposure of mainly male animals to chemical mutagens. This kind of testing does not identify chemicals producing only gene mutations or deletions and can therefore only provide positive evidence of mutagenicity and can never show that a chemical is not mutagenic.

Mikamo counted lethal mutations but did not study mutations in survivors. Nomura (1982) of the Institute for Cancer Research, Osaka University, used both X-rays and a chemical mutagen, urethane, on both male and female mice before mating and studied congenital malformations and tumours in survivors. Urethane is a potent chemical mutagen but nevertheless does not cause dominant lethal mutations or chromosomal abnormalities. Examination of 5,830 embryos by Nomura revealed no significant increase of dominant lethal mutations following dosing of a parent with urethane. X-rays on the contrary produced dominant lethal mutations as expected. Most malformations and tumours are, however, caused by gene mutations not by visible chromosomal abnormalities and urethane given to male or female mice before mating was very effective in producing malformations as shown in Figure 2.5 and tumours as shown in Figure 2.6. In one series of Nomura's experiments malformations were recorded in 19-day old foetuses, as shown in Figure 2.5., and in a second series in 7-day old offspring. The rate of malformations fell by 50 per cent between 19 day old foetuses and 7 day old pups because of the high mortality shortly after birth. At 7 days there were more than 10 times as many malformed pups as in controls. One of the less lethal malformations, open eyelid, was shown to be heritable. The tumours were diagnosed in offspring at 8 months of age and were found to be heritable in experiments confined to the male line as far as the F3 generation with a dominant pattern of inheritance and about 40 per cent penetrance. The strain of animals used may have had a particular susceptibility to tumour initiation. It is apparent, however, from these experiments that tumours in offspring can be produced by exposure of the parental germ line to mutagens before fertilization. The females were irradiated or dosed with urethane in what was described as the "late follicular stage" which covered the 14 days before ovulation. As there are profound changes in female susceptibility during these 14 days Nomura's studies do not help in the detailed description of the susceptible period.

While the highest susceptibility in women may only last about 41/2 days as suggested in Figure 2.4 animal experiments show that susceptibility, although at a lower level, increases from the beginning of the cycle, which in women is 14 days before ovulation and furthermore that susceptibility particularly to single gene mutations begins to increase earlier still as long as 40 days before mating in the mouse, equivalent to about 100 days in women (Russell, 1977). Russell in one series of experiments examined 258,663 mouse pups for 7 specific mutations following irradiation of the dams with 50 rads at a rate of 50 rads/min. No mutations were recorded for 92,059 pups with an interval between irradiation of their dams and mating greater than 42 days. There were 7 mutations in 71,070 pups following irradiation 21 to 42 days before mating. Animal studies suggest that the dormant mammalian female germ line may be insensitive to the action of most mutagens until a few weeks before ovulation when the period of increased susceptibility begins (Oakberg, 1979). Does the comparative insensitivity of dormant germ cells apply to women?

Susceptibility in women must build up to the high level around ovulation, but there is no direct evidence as to how long this takes. The beginning of susceptibility 100 days before ovulation is a cautious estimate based on the animal experiments. Cox and Lyon (1975), of the Radiobiology Unit at Harwell, discussing their experiments on guinea pigs and hamsters, said that it would be imprudent to assume that no mutations could be induced by radiation in the dormant, or immature oocyte, and they added that it is reasonable to assume only that it is more difficult to cause mutations in the dormant human oocyte than in the mature oocyte. They also suggested that the susceptibility of the human oocyte during the period prior to ovulation may not be very different from that found in experimental mammals. This is a cautious assumption for couples and those who advise them.

SUSCEPTIBLE PERIODS FOR THE ORIGIN OF DOWN'S SYNDROME

The importance of susceptible periods may be shown by examining the origins of a particular genetic disease caused by chromosomal abnormality, namely Down's syndrome. The knowledge of when Down's syndrome originates is part of the knowledge needed for its prevention. In Chapter One the role of new mutations in maintaining the prevalence of genetic diseases in any human population was discussed, and it was noted that Down's syndrome prevalence is maintained largely by new mutations, with an inherite0d predisposition in a small percentage of cases. Down's syndrome would disappear if it were not constantly renewed by new mutations. Between I and 2 in 1,000 newborns suffer from Down's syndrome. About three-quarters of all cases of Down's syndrome conceived are lost by miscarriage. Down's syndrome is somewhat unusual in that a quarter are carried to term, as most chromosomal abnormalities of the embryo result in miscarriage with no survivors (Bouet al., 1981a, 1981b).

The French Medical Research Council reported in 1970 that, using the characteristic markings of chromosomes, it was possible to say whether the extra chromosome in Down's syndrome had its origin at meiosis I or II (de Grouchy, 1970). Two years later two research workers at the Karolinska Hospital in Stockholm showed that the parental origin of the extra chromosome could be traced (Licznerski & Lindsten, 1972). In 1983 two paediatricians at the Children's Medical Center in Dayton, Ohio, summarized the results of 30 studies from Austria, Denmark, France, Netherlands and the U.S.A. Table 2.1 shows that of 369 cases of Down's syndrome 73.2 per cent originated at meiosis I and 26.8 per cent at meiosis II (Juberg & Mowrey, 1983). This is further evidence for the conclusion that Down's syndrome has its main origin in women during the 60 hours or so before ovulation. Juberg and Mowrey (1983) commented on the paternal responsibility for 20 per cent of cases:

"The fact that 20 per cent of the cases arose from spermatogenic nondisjunction has important implications. The first is that physicians and counsellors can help remove the onus that trisomy 21 syndrome is exclusively attributable to the mother. Second it should soon be possible to study a group of fathers for factors contributing to nondisjunction. Avoidance of an offending environmental agent might be possible after such studies.

TABLE 2.1

ORIGINS OF DOWN'S SYNDROME IN GERM CELLS OF MOTHER OR FATHER; 369 CASES FROM 30 STUDIES, EUROPE AND U.S.A.

meiotic	mother	father	both parents
division

	N	%	N	%	N	%
first	225	61.0	45	12.2	270	73.2
second	67	18.1	32	8.7	99	26.8
total	292	79.1	77	20.9	369	100.0

Source: Juberg & Mowrey, 1983

The suggestion that nondisjunction may be caused by an "offending environmental agent" is supported by reports that nondisjunction can be produced in animals by exposure to a variety of chemical compounds, about 20 having been listed by 1983 (Hansmann, 1984). A number of papers speculate as to whether nondisjunction is preceded by some genetic predisposition possibly caused by a mutation during the early production of the eggs in the female foetus. There is so far little evidence for such a predisposition, except in the 5 per cent or so of cases where the parent is carrying diagnosable chromosomal abnormalities. The animal experiments show moreover that nondisjunction can be produced at will by many different kinds of physical and chemical disturbmce during the active stages of meiosis without predisposition, and nondisanction is difficult to produce experimentally at any other time. Predisposition less it can be diagnosed also offers little preventive opportunity and the safer hypothesis for a couple is that nondisjunction is caused not long before happens during the highly susceptible periods around meiosis I and II. Chemicals that stop or slow down cell division, introducing a delay of a few hours, have been shown to increase the risk of nondisjunction in rat and Chinese hamster cells (Kamiguchi et al., 1979). Chemicals that slow down division in this way include fungicides, organic solvents, anaesthetics, anti-cancer drugs and anti-anxiety drugs (Hsu et al., 1983; Liang et al., 1983). Diazepam (valium) was used by Hsu to stop cell division temporarily in female hamster cells. An arrest of cell division of 2 hours just before the final stages of meiosis I did not apparently damage the cells, but a temporary arrest of 7 hours resulted in many cells at subsequent divisions having the wrong number of chromosomes. In vivo temporary arrest of meiosis I and II may be caused by depression of the hypothalamic gonadal axis, for example by psychotropic drugs including alcohol (Gavaler & Van Thiel, 1987). The effects of such drugs are, however, distributed throughout the axis and it has been shown that alcohol can produce aneuploidy in mouse eggs after in vivo and in vitro activation with alcohol (Kaufman, 1985). Down's syndrome is discussed again in Chapter Eight in the context of ageing.

Apart from Down's syndrome the commonest diseases caused by chromosomal abnormalities are those involving the sex chromosomes, which are also mainly caused by one-generation inheritance from mother or father (Sperling, 1984). Both male and female sex chromosomal abnormalities have a frequency between 1.0 and 1.5 per 1,000 births (Buckton, 1983). The commonest abnormalities are Klinefelter's syndrome in boys and Turner's syndrome in girls, both associated with infertility. In all about 6 newborns in 1,000 have a recognizable chromosomal abnormality of which about 3 in 1,000 have some clinical significance. Six to ten per cent of stillborn infants and 5 to 7 per cent of children who die in infancy or early childhood have been reported to have chromosomal abnormalities (Hook, 1982).

SUSCEPTIBLE PERIOD IN WOMEN FOR THE ORIGIN OF MISCARRIAGE

An inherited propensity for any characteristic that increases infertility is unlikely to be common as it must tend to die out quickly. Among 24,951 American women undergoing amniocentesis it was found that about 5 in 1,000 were carrying translocations that might cause miscarriage (Hook et al., 1984). A compilation of the cytogenetic findings of 79 published surveys of couples with two or more pregnancy losses showed an overall prevalence of chromosomal abnormalities of 2.8 per cent, a figure 5 or 6 times higher than the frequency in the general population of parents (Tharapel et al., 1985). The results are summarised in Table 2.2 where it is seen that 31 per cent of the aberrations were carried by the men and 69 per cent by the women. Insofar as this 2.8 per cent of the couples owed their reduced fertility to inheri0ted abnormal chromosomes the abnormalities must have been largely the result of new mutations in the germ cells of their own parents. In over 97.2 per cent of cases there was no evidence that the propensity was inherited by the couples who suffered the miscarriages.

Most miscarriages are a consequence of the fertilized ovum being defective. There have been a number of cytogenetic studies of aborted embryos. Carr (1970) discussed the origin of many miscarriages in chromosomal abnormalities. Boue and Boue (1976) reported that 66 per cent of early spontaneous miscarriages before 8 weeks gestation had such chromosomal abnormalities. Poland et al. (1981) found 84 per cent of miscarried embryos less than 3mm long were abnormal and 57 per cent when karyotyped were found to be chromosomally abnormal. The data on some 5,000 miscarried embryos had been reported by 1981, and it is apparent from the chromosomal abnormalities alone that most of the defects in ova leading to miscarriage are already present in the zygotes immediately after fertilization and that they have their origin in male or female germ cells before fertilization. Most miscarriages of female origin begin during ovulatory maturation and an error at meiosis I, most frequently a nondisjunction, is the commonest cause of miscarriage.

TABLE 2.2

AFTER TWO OR MORE PREGNANCY LOSSES; 79 STUDIES

	women	men	total
number	8,208	7,834	16,042
number with abnormalities	319	143	362
per cent with abnormalities	3.9	1.8	2.8

Attempts have been made to prevent early miscarriage using hormones such as progesterone and drugs in early pregnancy. If in most cases of miscarriage the embryo is defective and the defects are already present in the zygote there is a danger that preventing miscarriage after fertilization will salvage defective embryos and increase the risk of a newborn with birth defects. Only intervention before conception, and, indeed, before ovulation can succeed in the primary prevention of miscarriage.

Hertig, an American pathologist at the Boston Lying-in Hospital, reported in the 1940s on 1,000 miscarriages and concluded that 62 per cent were connected with defects of the human ovum (Hertig & Sheldon, 1943). Twenty years later he published data indicating that these defects of the ovum had their origin before ovulation and were commoner if ovulation had been delayed (Hertig, 1967). He said in one of his papers that if in women the ovum lingers longer in the follicle than day 14 it has an increasing chance of becoming a "defective ovum" when fertilized. The two lower rows in Figure 2.7 use Hertig's data and show the day of ovulation of normal and abnormal ova.

The average time of ovulation of normal ova was 14 days and of abnormal ova 17 days. The upper row in Figure 2.7 shows the time of ovulation for pregnancies terminating in miscarriage (Iffy, 1981). The series of cases used to produce the upper row actually showed that premature ovulations as well as delayed ovulations were associated with miscarriages but the delayed ovulations were commoner and appeared to be the greater risk. Evidence of the apparently damaging effect of delay in ovulation in women inspired animal experiments and several teams of investigators in different countries found that delay in ovulation increased the risk of chromosomal abnormalities (Bomsel-Helmreich et al., 1979; Butcher & Fugo, 1967; Kamiguchi et al. 1979; Mikamo & Hamaguchi, 1975). New mutations were produced experimentally not by any direct poisoning of the ovum but by upsetting its hormone supplies during development. Butcher (1981) showed that a delay in ovulation for 48 hours in female rats caused a range of congenital malformations at every stage of embryonic and foetal development. Most of these malformations were not compatible with life but a small percentage were, and included abnormalities of the neural tube like spina bifida. The serious consequences of a delay of ovulation were similar whether or not the delay was caused by using drugs. If anything delays the LH surge meiosis and ovulation are delayed. It is seen in Figure 2.8 that in rat dams a low dose of smoke delayed the LH surge by about 1 hour

and a high dose by about 2 hours (McLean et al., 1977). The hypothalamus can delay or inhibit the LH surge. Nicotine in tobacco smoke is an example of many substances which act on the hypothalamus causing this delay. It is seen in Figure 2.9 that 3 doses of nicotine caused both a 2 hour delay in the LH surge and a depression to about half the normal level when injected into rat dams shortly before the normal time of the LH surge (Blake et al., 1972). Four doses resulted in 5 hours' delay and a depression to only about a quarter of the normal level. High doses of a poison like nicotine stop the LH surge altogether so the meiosis and ovulation stop and reproduction is prevented.

Drugs which affect the brain, including tranquillizers, narcotics, hypnotics, sedatives and alcohol, quite generally affect the secretion of sex hormones acting through the hypothalmus and pituitary glands (Smith, 1983)> The exposure may not be severe enough to cause the hypothalmus to stop reproduction, but it may so affect follicular development as to prejudice the outcome after fertilization. Follicles, considering only size, can be either more or less grown. Follicles less than 16 mm in diameter at the time of ovulation are liable to die and disappear without producing a satisfactory, viable ovum. The normal range is 22 to 30 mm in diameter (Muller-Tyl et al., 1984). The follicle has to reach a certain size for the ovum to survive. Less than satisfactory development of the follicle can prejudice subsequent development of the ovum (Bomsel-Helmreich et al., 1979; Jongbloet, 1986). The granulosa cells produced during follicular development may be too few in number, too small or otherwise defective and produce an inadequate corpus luteum (Dizerega & Hodgen, 1981). After ovulation the granulosa cells stop dividing so the size of the corpus luteum is decided before ovulation. If the follicle is too small so is the corpus luteum.

Failure of the corpus luteum to provide an adequate supply of hormones, particularly progesterone but also oestrogens, is a mechanism of miscarriage following faulty follicular development because these hormones are essential to the first 6 to 7 weeks of pregnancy (Heap & Flint, 1984). If the hormones fail the pregnancy fails. During the first 8 weeks the placenta gradually takes over the task of supplying the hormones needed to maintain pregnancy and the corpus luteum is no longer needed from about the beginning of the third month of gestation. Anything that delays or depresses the supply of these pregnancy hormones is likely therefore to cause miscarriage. Smoking and exposure to nicotine would for this reason alone be expected to cause miscarriage as, indeed, it does, as shown in Figure 2.10 from a survey of smoking habits of women attending a New York hospital and their miscarriages compared to non-smokers.

The development of the embryo and placenta depends upon a blood supply containing adequate concentrations of hormones and nutrients including progesterone and oestrogens provided by the supply of hormones by the corpus luteum can slow down DNA synthesis and cell replication in the embryo. During the early stages of embryonic development the corpus luteum is also still dependent on luteotrophic hormones from the pituitary, so that anything that depresses the hypothalamic pituitary axis may also depress embryonic growth. However as the size of the corpus luteum is wholly determined during follicular development it is apparent that embryonic development has in some measure been already programmed before ovulation during growth of the follicle. As most chromosomal abnormalities have their origin at meiosis I before ovulation, and congenital malformations can be produced in animals by slowing down the growth of the follicle, the importance of normality of follicular development is apparent. The growth of the follicle is illustrated in Figure 2.11. The normal human follicle increases in diameter 12.5 times and in mass about 2,000 times in 14 days. This is the highest rate of growth found at any time during the human life cycle. Cell numbers in the follicle have to double about 11 times in 14 days, doubling once about every 30 hours. This is only possible if the follicle has a supply of blood with the right concentrations of hormones and nutrients and no poisons.

A high rate of follicular growth requires a high rate of synthesis of DNA. One cause of unsatisfactory pregnancy is a slow-down in rates of DNA synthesis beginning during ovulatory maturation in the female and extending through fertilization and embryonic development. The association of reduced mass of DNA in congenitally malformed pups and their placentas compared with normal pups is illustrated in Figures 2.12 & 2.13, based upon research by Potier de Courcy and colleagues at the French national laboratory for nutrition research (CNRS).

The livers of the mothers of the malformed pups and their placentas had reduced DNA as shown in Figure 2.12. In this example the mothers were deprived of pantothenic acid and the foetal brain is seen to have been spared compared with the whole foetus or foetal liver, but neither foetus nor placenta were spared compared with the matemal liver (Potier de Courcy, 1966). Figure 2.13 shows the DNA content of malformed foetuses produced by riboflavin deficiency; the foetal brain is seen to have been spared compared with the maternal liver and indeed with the whole foetus and placenta (Potier de Courcy et al., 1970; Potier de Courcy et Terroine, 1968). In Figure 2.13 the deprivation only began at mating while in Figure 2.12 it began 14 to 10 days before mating. Different deficiencies with very different time-scales are seen to produce different patterns of slow-dowr DNA synthesis.

MOTHER, DAUGHTER, GRANDDAUGHTER

Susceptibility in the cycle of generations is shown diagrammatically in Figure 2.14. In the interests of simplicity this figure simplifies, some people may say oversimplifies, language. Twenty-four days after conception new ova are shown in this figure to be visable. These new ova are the cells that carry the genetic code from one generation to the next, the germ cells. In a female embryo there are only around 100 of these new germ cells in the baby daughter's ova in Figure 2.14, 24 days after conception, but their number doubles about every 10 days to reach a maximum of about 7 million around the 6th month of gestation. Their number then begins to decline and continues to do so for the rest of the daughter's life so that few remain when she reaches 50. Of the 7 million new ova only one is needed for a mother eventually to have a granddaughter and a second to have a grandson arid so for the next generation to be reproduced.

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