Effects of maternal caffeine consumption on ovarian follicle development in wistar rats offspring.

BACKGROUND: In recent years concerns have been raised about human reproductive disorders, specially the effects of environmental factors on human fertility and pregnancy outcome. Therefore, the present study was designed to assess the effects of maternal caffeine consumption on ovarian follicles development in rat offspring.
METHODS: 60 pregnant female rats were randomly divided into a control and two experimental groups. The rats in the two experimental groups received caffeine via drinking water during gestation (26 and 45 mg/kg) and lactation (25 and 35 mg/kg). The ovaries of the offspring were removed at 7, 14, 28, 60, 90 and 120 days after birth, and fixed in Bouin's solution. By preparing serial tissue sections, structural changes in ovarian follicles and corpora lutea were studied during postnatal development.
RESULTS: The weight of ovaries decreased significantly (p<0.05) in the high dose caffeine-treated group at all stages of postnatal development. Significant (p<0.05) decreases were seen in the number of primordial follicles from day 7 to 120 after birth in the high dose caffeine-treated group. Moreover, the number of primary and secondary follicles decreased significantly on days 7, 14 and 28 as did the number of antral follicles on days 14 and 28 after birth (p<0.05) in the high dose caffeine-treated group. The diameter of secondary and antral follicles decreased significantly (p<0.05) in high dose caffeine-treated group on the early days of postnatal development. No statistically significant differences were seen in the number of corpora lutea between the groups.
CONCLUSION: The present study shows that caffeine consumption during gestation and lactation affects the early stages of ovarian follicle development and reduces reproductive efficiency in the offspring of Wistar rats.

Background

In recent years, concerns have been raised regarding the increases in human reproductive disorders. Exposure to environmental and occupational toxicants and progressive changes in many aspects of lifestyle, including dietary habits, has been shown to deteriorate reproductive health, thus, affecting the ability of couples to conceive and maintain a healthy pregnancy (1).

Among dietary factors, caffeine consumption has been found to adversely affect reproduction (2). Caffeine is a naturally occurring alkaloid found in the seeds, leaves and fruits of more than 60 plants such as in coffee and cocoa beans, kola nuts, and tea leaves. Caffeine is present in a wide variety of foods and beverages including coffee, tea, cocoa, chocolate, colas and energy drinks, and also in a number of prescription drugs including cold and flu remedies, headache treatments, diet pills, diuretics and stimulants (3). The amount of caffeine in foods and beverages varies widely depending on the type of product and the preparation methods. In general, brewed coffee contains the highest amount of caffeine, with an average of 137 mg per an-8-ounce cup and chocolate contains the lowest amount of caffeine (4). The total daily intake of caffeine from all sources is estimated to be about 3-7 mg/kg per day or 200 mg/day (5). Moreover, it has been shown that caffeine in shampoos is absorbed through the skin, mostly through hair follicles, and reaches the blood stream (6).

Peak plasma caffeine concentration is reached between 15-120 min after oral ingestion in humans. Caffeine half-life is about three hours but it increases to 5.6 hours in the first trimester and to 18 hours in the 35th week of pregnancy (7). Caffeine half-life increases to 80 hours during neonatal period due to the decreased activity of cytochrome P-450 (8). Caffeine crosses the placenta easily and is found in both the fetus and in newborn infants (9).

Caffeine consumption has been found to be associated with low birth weight, small head, cleft palate, retardation, miscarriage or spontaneous abortion, increased incidences of digital defects and delayed ossification (9, 10). Wilcox et al. found a 50% reduction in the probability of conception per cycle with tintake of one cup of coffee per day (11). Jagilo et al. reported that subcutaneous administration of 25 mg/kg per day of caffeine prevented hormonally induced superovulation in adult mice (12). Similarly, it was found that oral gavage of 136 mg/kg per day of caffeine, 5 days per week for a total of 100 days, blocks normal oogenesis in adult rats (13). Furthermore, it has been shown that caffeine prolongs the meiotic arrest of porcine oocytes at the germinal vesicle stage (14). However, epidemiological studies in Danish women showed no association between delayed conception and consumption of caffeinated beverages (15, 16). Likewise, Caan et al. found that women who drank more than half a cup of tea per day had a significant increase in fertility (17). Furthermore, caffeine administeration during pregnancy did not affect the rate of early mitotic proliferation of germ cells at day 20 of pregnancy in rats, nor did it prevent their development or decrease their meiosis (18). Caffeine treatment can prevent the decline in the developmental potential of ovine oocytes matured and aged in vitro (19).

Regarding the conflicting results of epidemicological studies on caffeine and its effects on reproductive outcomes (20) and caffeine-containing foods and beverages are so widely consumed by most human populations of the world, its health effects have been and are still being studied extensively. The present study was designed to investigate the effects of maternal caffeine consumption during gestation and lactation on postnatal development of ovaries in the offspring of Wistar rats.

Methods

Animals

Wistar rats were obtained from animal house of Jondishapour Medical Sciences University of Ahwaz and kept under specific conditions on a constant 12-hour light/dark cycle and at a controlled temperature of 22 ± 2° C. Standard pellet food and distilled water were available ad libitum. Female Wistar rats (100 ± 10 days old) were mated overnight at a 3:1 females:male ratio. Vaginal smears were daily prepared and examined for the presence of sperm. The day of sperm detection in vaginal smears was considered day 0 of pregnancy. 60 Pregnant female Wistar rats were randomly divided into three equal groups; a control group and two treatment groups. The two treatment groups, respectively received caffeine via drinking water during gestation (26 and 45 mg/kg) and lactation (25 and 35 mg/kg) (23, 24) i.e. until day 21 after birth. The doses were established from related reproductive and developmental studies. On days 7, 14, 28, 60, 90 and 120 five pups were randomly selected, weighed and their ovaries were removed under chloroform inhalation anesthesia. The ovaries were trimmed of fat and extraneous tissue, weighed and fixed by immersion in Bouin's solution for 24 hours.

Microscopic Study

Following tissue processing, 5 µm serial paraffin sections were prepared and stained by haematoxylin-eosin. For microscopic examination, sections were selected using a non-random 10% sampling method and ovarian follicles and corpora lutea were counted in every 10th section of the ovary (25), in a fashion that each counted section was separated by a distance of approximately 50-60 μm from the next 10th section.

Later, the mean number of ovarian follicles and corpora lutea of each ovary were calculated in the selected sections. Differential follicle counting and categorizing were performed blindedly. Ovarian follicles were classified on the basis of ovarian follicle morphology. Follicles containing a single layer of squamous follicular cells were considered as primordial. A primary follicle contains an oocyte surrounded by a single layer of cuboidal follicular cells; the secondary follicle contains more than one layer of follicular cells around the oocyte. Follicles containing scattered spaces or a distinct antrum were considered as antral (26). All the follicles were classified as either healthy or atretic, respectively, according to the absence or presence of signs of oocyte and/or granular degeneration such as pyknosis of the nucleus, infolding of the cell wall in oocyte, ingression of granulosa cells within the antral cavity, pulling away of granulosa cells from the basement membrane, infolding or thickening of base membrane and uneven layers of granulosa cells (27).

For measuring the diameter of ovarian follicle in each developmental stage, 45 microscopic fields were randomly chosen in each rat. Then, using the ocular micrometer of a light microscope (Olympus EH, America Inc.), at a magnification of 10×, the diameter of each ovarian follicle were measured. To avoid recounting the same follicle, only individual follicles, containing an oocyte with a nucleus, were evaluated.

Statistical Analysis

The data were analyzed using SPSS, version 10.0 for Windows. Body and ovarian weights, the number and diameter of follicles and corpora lutea in the control and caffeine-treated groups were compared by one-way analysis of variance (ANOVA) and Tukey's post hoc test. Comparisons between different groups were made in each developmental stage, independent from other days of postnatal development. The differences were considered to be significant when p<0.05.

Results

Mean body weight showed significant (p<0.05) decreases in high dose caffeine-treated group in all stages of postnatal development in comparison with the control group. There were significant (p<0.05) differences between the mean ovary weight in high dose caffeine-treated and control groups in all stages of postnatal development. No significant differences were seen between the mean body and ovary weight in low dose caffeine-treated and the control groups in all stages of postnatal development (Table 1).

Table 1

Distribution of body weight (gr) and ovary weight (mg) in the controls and caffeine-treated offspring of Wistar rats during different stages of postnatal development (Mean±SEM)

Parameters	Age of Pups (day)	Groups
		Control	Caffeine-treated
			Low dose	High dose
Body weight (gr)
	7	8.91±0.17a	7.84±0.41a,b	7.02±0.30b
	14	12.14±0.76a	11.35±0.28a,b	9.86±0.38b
	28	25.00±1.18a	22.75±1.21a,b	20.93±1.25b
	60	58.17±3.62a	52.96±2.89a,b	50.96±2.78b
	90	82.61±4.18a	77.14±3.62a,b	73.79±3.59b
	120	95.12±4.26a	91.65±4.01a,b	88.05±4.11b
Ovary weight (mg)
	7	2.20±0.15a	1.80±0.18a,b	1.20±0.20b
	14	6.30±0.20a	5.80±0.31a,b	5.20±0.41b
	28	18.40±1.33a	17.50±1.24a,b	15.80±1.03b
	60	35.00±2.32a	32.70±2.22a,b	30.60±2.17b
	90	45.61±3.54a	40.90±3.44a,b	38.70±3.28b
	120	48.33±5.01a	46.60±4.41a,b	42.21±4.12b

Different superscripts in the same row indicate significant difference between groups (P<0.05)

The mean number of primordial follicles decreased significantly (p<0.05) in high dose caffeine-treated group from the 7th to 120th day in comparison with the control group (Table 2).

Table 2

The number of ovarian follicles in the control and caffeine-treated offspring of Wistar rats during different stages of postnatal development (Mean±SEM)

Follicular stage	Age of pups (days)	Groups
		Control	Caffeine-treated
			Low dose	High dose
Primordial F.
	7	48.57±5.29a	44.57±4.27a,b	39.57±5.27b
	14	38.70±4.72a	36.40±4.27a,b	32.10±3.27b
	28	25.50±3.14a	21.20±2.25a,b	17.10±1.36b
	60	19.00±1.67a	16.33±1.21a,b	11.41±1.28b
	90	17.66±1.46a	12.73±1.18a,b	10.33±1.16b
	120	15.32±1.25a	13.87±1.20a,b	10.65±1.04b
Primary F.
	7	35.90±4.97a	28.10±3.21b*	25.90±2.39b**
	14	30.40±3.27a	22.90±2.27b*	20.40±2.13b**
	28	18.08±2.25a	9.16±1.18b*	8.75±1.11b**
	60	8.26±1.21	6.20±0.27	5.80±0.22
	90	8.40±1.38	5.13±0.24	6.53±0.62
	120	6.43±0.03	6.30±0.25	5.05±0.23
Secondary F.
	7	3.00±0.15a	1.90±0.11b*	1.50±0.11b**
	14	16.80±1.45a	10.70±1.28b*	9.00±1.15b**
	28	11.75±1.41a	5.75±0.22b*	4.00±0.22b**
	60	8.10±1.20	6.30±0.28	5.90±0.19
	90	3.93±0.15	3.50±0.14	3.44±0.14
	120	4.64±0.17	3.55±0.18	3.84±0.16
Antral F.
	7	N	N	N
	14	3.62±0.15a	1.25±0.10b*	1.04±0.10b**
	28	4.83±0.18a	2.04±0.12b*	2.00±0.12b**
	60	4.80±0.17	3.30±0.15	3.20±0.16
	90	5.07±0.28	4.41±0.21	4.23±0.18
	120	7.65±0.41	6.71±0.30	6.46±0.28

Different superscripts in the same row indicate significant difference between groups (P<0.05)

p<0.01

p<0.01

F: Follicle, N: Not seen

Significant (p<0.05) decreases were observed in the mean number of primary and secondary follicles on days 7, 14 and 28 in both treatment groups in comparison with the controls. There were no significant differences between the mean number of primary and secondary follicles in the controls and both treatment groups on days 60, 90 and 120 (Table 2).

Antral follicles were not seen on day 7 in any of the experiment groups. The mean number of antral follicles decreased significantly (p<0.05) in both treatment groups on days 14 and 28 in comparison with the control group (Table 2). No significant differences were seen between the mean number of antral follicles in the control and both treatment groups on days 60, 90 and 120 (Table 2).

There were no significant differences between the means of primordial and primary follicles in diameter in the control and treatment groups in all stages of postnatal development. The mean for secondary follicles diameter decreased significantly (p<0.05) in high dose caffeine-treated group on days 7, 14, 28 and 60 in comparison with the controls. Moreover, significant (p<0.05) decreases were seen between the mean of antral follicle diameter in the control and high dose caffeine-treated group on the 14th, 28th and 60th days (Table 3).

Table 3

The diameter of ovarian follicles (µm) in control and caffeine-treated offspring of Wistar rats during different stages of postnatal development (Mean±SEM)

Follicular stage	Age of pups (days)	Groups
		Control	Caffeine-treated
			Low dose	High dose
Primordial F.
	7	19.50±1.80	19.30±2.41	18.90±1.08
	14	20.25±2.85	19.59±1.82	19.30±1.32
	28	21.60±3.10	20.92±2.73	20.25±2.36
	60	21.35±3.83	20.74±2.55	20.79±1.47
	90	20.90±2.90	20.25±1.18	20.43±2.16
	120	20.74±2.45	20.32±2.20	20.45±2.24
Primary F.
	7	45.29±2.28	44.74±2.20	44.30±2.10
	14	50.24±3.26	49.73±2.67	48.55±2.30
	28	50.30±3.31	50.25±3.28	49.58±2.03
	60	51.55±2.01	50.67±3.39	50.38±2.33
	90	51.80±3.84	51.35±2.53	50.61±3.36
	120	51.84±2.86	51.40±2.57	50.80±3.41
Secondary F.
	7	70.10±3.27a	68.20±4.10a,b	61.30±3.87b
	14	88.72±4.70a	83.59±3.47a,b	78.55±4.31b
	28	109.89±4.41a	100.81±4.05a,b	94.65±3.34b
	60	112.93±5.47a	106.88±5.49a,b	99.37±4.83b
	90	116.39±5.56	112.32±4.61	110.33±3.26
	120	117.65±4.60	114.71±5.10	111.46±5.39
Antral F.
	7	N	N	N
	14	200.00±6.12a	196.62±4.47a,b	190.15±4.67b
	28	201.90±5.60a	199.05±5.25a,b	192.74±5.10b
	60	205.09±6.03a	201.31±6.42a,b	195.17±4.80b
	90	220.80±5.22	218.55±5.11	215.23±6.21
	120	222.65±5.80	220.71±6.16	217.42±5.33

Different superscripts in the same row indicate significant difference between groups (P<0.05)

F: Follicle, N: Not seen

There were significant (p<0.05) increases in the mean number of atretic follicles on days 7, 14 and 28 in both treatment groups in comparison with the controls. No statistically significant differences were seen between the mean number of corpora lutea in the controls and both treatment groups on the 60th, 90th and 120th days.

Discussion

The aim of present study was to establish whether in utero and lactational exposure of Wistar rats to caffeine affects ovarian development in their offspring during pre- and post pubertal development. The results clearly showed that high dose maternal caffeine consumption during gestation and lactation significantly reduced ovarian weight and primordial follicle population and diminished fertility and reproductive efficiency in the offspring of Wistar rats. These alterations in the ovary were associated with a significant growth retardation of the female off-spring. It seems that reductions in the number of primordial follicles, the number and diameter of secondary and antral follicles and the increase in the number of atretic follicles caused a significant decrease in ovarian weight in different stages of postnatal development.

In this study, reduction in number of primordial, primary and secondary follicles increased from birth to 28 days on wards but decreased then after, up to the end of the study (120th day) in both treatment groups. However, the highest ovarian weight decrease was seen on day 7 after birth which contiuned until the 120th day in both treatment groups.

Female fertility depends on the supply and maturation of the ovarian germ cells, i.e., the oocytes and the differentiation and proliferation of ovarian somatic cells, or granulosa and thecal cells (28). During folliculogenesis, oocytes grow and they are surrounded by an increasing number of granulosa cell layers. From the preantral stage onward, theca cells differentiate outside the follicles (29). Improper ovarian differentiation or folliculogenesis, due to intra- or extra-ovarian regulation defects, often results in premature ovarian failure, leading to infertility (30). In rats, the formation of primordial follicles is completed by postnatal day three or four (31). Most primor-dial follicles remain quiescent, but some grow and undergo transition into the primary follicle stage. High numbers of preantral and antral follicles would be present from day 9 to 20 (32), and the first estrus and ovulation will occur between days 35-42 after birth (33).

The present study showed that caffeine treatment altered the number of primordial follicles in the resting pool from day 7 until day 120 in the high dose caffeine group.

For reproductive toxicants that cause direct damage to ovarian follicles, the stage of development at which follicles are destroyed determines the impact that exposure to the chemical will have on reproduction (34). Chemicals that extensively destroy primordial follicles cause permanent infertility and premature ovarian failure, since once a primordial follicle is destroyed, it cannot be replaced. Destruction of primordial follicles will have an irreversible effect on reproduction until such a time that recruitment for the number of growing and antral follicles can no longer be supported (35, 36). It seems that caffeine could interfere with cell division resulting in decreased number of cells and cell death. Axelrod and Reichenthal described this hypothesis that caffeine acts by genetic damage to the cells replicating DNA (37). Comet assay studies indicate that caffeine-mediated increases in radiation risk of embryos is due to the inhibition of DNA repair (38). Furthermore, Jagiello et al. (1972) reported meiosis suppression by caffeine in female mice (9).

However, our findings indicated that unlike days 7, 14 and 28, there were no significant differences in the number of growing follicles on days 60, 90 and 120 after birth in the treatment groups. More-over, the diameter of secondary and antral follicles and the number of corpora lutea in the treatment groups did not differ with that of the control group on days 60, 90 and 120 after birth. Thus, it seems that caffeine exposure has transient effects on follicular development and ovulation in the offspring of Wistar rats during the postnatal development. This could be due to the fact that caffeine does not accumulate in the body over the course of time and it is normally excreted within several hours of consumption. Chemicals that selectively damage large growing or antral follicles only temporarily interrupt reproductive function because these follicles can be replaced by recruitment from the greater pool of primordial follicles. Thus, these chemicals produce a readily reversible form of infertility that is manifested relatively soon after exposure period (35, 36).

Conclusion

The present study showed that maternal caffeine consumption during gestation and lactation affects the early stages of follicular development and reduces reproductive efficiency in the offspring of Wistar rats.