Effect of long‐term caffeine administration to mice on in vitro fertilization and embryo development using oocytes

Abstract

Purpose

To evaluate the effect of long‐term caffeine administration to mice on in vitro fertilization (IVF) of oocytes.

Methods

Mice were injected with different dosages (0, 0.1, and 1.0 mg/mouse/converted day) of caffeine for one month. Subsequently, the fertilization rate and embryo development to blastocyst stage were evaluated in IVF using oocytes from the mice.

Results

The retrieved average oocyte rate was significantly lower (27.4) in mice injected with 1.0 mg caffeine than in the control group (36.5; P < 0.05); the fertilization rate was significantly different between the 0 mg (317/401; 79.1 %) and 1.0 mg group (199/301; 66.1 %) (P < 0.05). At 96 h after insemination, the blastocyst formation rate was significantly decreased in the 1.0 mg group (94/199; 47.2 %) compared with the control (0 mg) group (237/317; 74.8 %) and 0.1 mg group (226/323; 70 %) (P < 0.05). When 1.0 mg caffeine was administered for two weeks, embryo development was significantly impacted.

Conclusions

Our findings suggest that caffeine administration negatively impacts oocytogenesis and embryonic development after IVF.

Introduction

In recent years, the pregnancy rate via assisted reproductive technology (ART) has been increasing for severely infertile women; however, even when normal patients without any specific abnormalities are treated with ART, many women do not achieve a successful pregnancy. One of the main infertility factors is advanced age. Furthermore, we are influenced by different environmental factors including food, pollution, and so on. There may be possible effects of common habitual caffeine ingestion on fertility among women.

Caffeine is one of the most widely and routinely consumed pharmacologically active substances and is, in general, considered quite harmless to human beings [1, 2].

Furthermore, caffeine consumption is associated with benefits such as a decreased risk of endometrial cancer [3] and colorectal cancer [4]; however, women who habitually ingest caffeine have been found to have increased rates of spontaneous abortion, stillbirth, and low‐birth‐weight infants [5, 6, 7, 8, 9]. The literature contains only a handful of reports addressing the effect of caffeine intake on fecundity [10, 11]. In our previous pilot study (in preparation for submission), daily coffee intake among patients undergoing ART treatment was found to adversely impact treatment outcome.

In rodents, it has been reported that the gestational length increased and the birth weight decreased with regular caffeine ingestion [12, 13, 14]; however, these studies did not address the relationship between caffeine intake and reproductive organ/pharmacological effects. The literature contains only a few reports addressing the relationship of caffeine and IVF in mice. Therefore, in the present study, we attempted to clarify whether retrieved oocytes from mice that had been administrated caffeine were negatively impacted for IVF in a similar manner as human oocytes.

Materials and methods

Caffeine treatment of mice

Caffeine (trimethyl‐13C3, 99 % pure) was purchased from Wako Chemical Industries (Osaka, Japan). Caffeine was dissolved into 0 mg/0.1 ml, 0.2 mg/0.1 ml, and 2.0 mg/0.1 ml solutions, which were divided into multiple tubes and stored at −20 °C. Prior to their use every other day, tubes were thawed at room temperature before injection. The treatment consisted of intraperitoneal injection of 0.1 ml every other day. Thus, a 2‐day dose was injected once for each concentration, resulting in an evaluation of 0, 0.1, and 1.0 mg per mouse per day.

First experiment

The objective of this experiment was evaluation of the effect on IVF using retrieved oocytes from mice that were subjected to long‐term caffeine administration. We first determined the critical dose point that significantly influenced the number of oocytes and embryo development in in vitro culture after IVF using oocytes retrieved from mice that had been administrated caffeine.

Mature Jcl:ICR strain 8‐week‐old female mice received an intraperitoneal injection of caffeine every other day (two‐day dose per injection) for one month. The mice were divided into three groups (11 mice in each group, 33 mice in total) based on caffeine dosage: 0 mg/mouse (control), 0.1 mg/mouse/converted day, and 1.0 mg/mouse/converted day (as described in “Caffeine treatment of mice” section). Each caffeine dose was injected on the terminal preparation day; subsequently, superovulation was induced via intraperitoneal injection of 10 IU pregnant mare serum gonadotropin (PMSG), followed 48 h later by administration of 5 IU human chorionic gonadotropin (hCG). Oocytes were collected 14 h later; subsequently, oocytes surrounded by a cumulus complex were inseminated with mouse sperm in HFF99 (Fuso Pharmaceutical Industries, Osaka, Japan), supplemented with 10 % synthetic serum substance (SSS; Irvine Scientific, Santa Ana, CA, USA). After fertilization, these embryos were incubated in HFF99 at 37 °C under 5 % CO₂, 5 % O₂, and 90 % N₂ until blastocyst formation or hatching.

Second experiment

The objective of this experiment was to evaluate the impact upon embryo development in IVF of oocytes retrieved from mice that had received 1.0 mg caffeine per mouse per converted day.

We assessed whether the same effects that we found during the first experiment recurred during a treatment interval of 0, 2, 4, and 8 weeks in each group of four mice; the zygote embryos were cultured in HFF99 at 37° C under 5 % CO₂, 5 % O₂, and 90 % N₂ until blastocyst formation or hatching.

Statistical analysis of the different groups was conducted by the chi‐square test. P < 0.05 was considered statistically significant.

We obtained certification for the mouse study from the institutional review board of our hospital.

Results

Following caffeine injection, mean ± standard deviation body weights were 39.0 ± 3.0 g, 36.7 ± 2.6 g, and 36.7 ± 2.6 g in the control (0 mg), 0.1 mg/day, and 1.0 mg/day groups, respectively. No significant differences were observed.

The number of normal ovulated oocytes decreased with increased caffeine dosage; the average number of normal ovulated oocytes per mouse in the 1.0 mg caffeine/day group was significantly lower (27.4) than that of the control group (36.5) (P < 0.05; Table 1). Harvested oocytes were used for the in vitro fertilization test; the fertilization rate was 317/401 (79.1 %) at 0 mg, 323/377 (85.7 %) at 0.1 mg, and 199/301 (66.1 %) at 1.0 mg. The fertilization rate was significantly less in the 1.0 mg group compared with the 0 mg group (P < 0.05); however, the two cell division rates were similar among the three groups. In regard to blastocyst formation at 96 h, the blastocyst formation rate was significantly decreased in the 1.0 mg group [94/199 (47.2 %)] compared with the control (0 mg) group [237/317 (74.8 %)] (P < 0.05). Also, the blastocyst formation rate was significantly decreased in the 1.0 mg group (94/199; 47.2 %) compared with the 0.1 mg group [226/323 (70 %)] (P < 0.05). Furthermore, the blastocyst formation rate 120 h after insemination was significantly decreased in the 1.0 mg group (121/199; 60.8 %) compared with the control group (258/317; 81.4 %) (P < 0.05; Table 2). Furthermore, blastocyst formation was influenced by caffeine administration of 1.0 mg per mouse per day during two weeks (Table 3).

Table 1.

Number of retrieved normally ovulated oocytes per mouse and body weight with different caffeine doses

Caffeine dose (mg/mouse) (mg)	Average number of normal oocytes/mouse	Average body weight (g)
0	36.5 ± 3.0a	39.0 ± 3.0
0.1	35.9 ± 9.3	36.7 ± 2.6
1.0	27.4 ± 6.4b	36.7 ± 2.6

^a–b P < 0.05

Table 2.

In vitro fertilization results and blastocyst formation with different caffeine dosages in mice

Caffeine dosage (mg/day):	0	0.1	1
Number of normal oocytes after insemination	401	377	301
Number of fertilized oocytes 2PN formation (%)	317 (79.1 %)a	323 (85.7 %)	199 (66.1 %)b
Number of 2 cell division oocytes (%)	317 (100.0 %)	321 (99.4 %)	198 (99.5 %)
Blastocyst formation 96 h after insemination	237 (74.8 %)c	226 (70.0 %)d	94 (47.2 %)e
Blastocyst formation 120 h after insemination	258 (81.4 %)f	246 (76.2 %)g	121 (60.8 %)h

^a–b P < 0.05, ^{c–e, d–e} P < 0.05, ^{f–h, g–h} P < 0.05

Table 3.

In vitro fertilization results and blastocyst formation with different durations of caffeine treatment

Caffeine treatment duration (1.0 mg/day)	0 weeks	2 weeks	4 weeks	8 weeks
Number of normal oocytes after insemination	112	108	78	90
Number of fertilized oocytes 2PN formation (%)	97/112 (86.6 %)	97/108 (89.8 %)	60/78 (76.9 %)	80/90 (88.9 %)
Number of 2 cell division oocytes (%)	97/97 (100 %)	97/97 (100 %)	60/60 (100 %)	79/80 (98.8 %)
Blastocyst formation 96 h after insemination	86/97 (88.7 %)a	73/97 (75.3 %)b	44/60 (73.3 %)c	52/80 (65.0 %)d
Blastocyst formation 120 h after insemination	89/97 (91.8 %)e	83/97 (85.6 %)f	47/60 (78.3 %)g	59/80 (73.8 %)h

^a–b P < 0.05, ^a–c P < 0.01, ^a–d P < 0.01, ^e–g P < 0.05, ^f–h P < 0.01, ^e–h P < 0.05

Discussion

We found that the number of retrieved oocytes was significantly decreased by administration of caffeine at 1.0 mg/day for more than two weeks. When retrieved oocytes were used for IVF, the blastocyst formation rate was significantly decreased in the 1.0 mg/mouse treatment group (mouse strain Jcl:ICR). When we began this caffeine study, we evaluated the effect of 0, 0.06, and 0.6 mg per day per mouse using a different mouse strain: B6D2F1. A decreased number of oocytes was found in the 0.6 mg group; however, embryo development was similar in the three groups (data not shown). According to these results, we thought it might be more appropriate to use a caffeine dose >0.6 mg to evaluate embryo development. We wanted to determine the critical dosage level, and plan future mouse studies with different dosages. The ICR strain is less expensive than the B6D2F1 strain; therefore, we switched the rodent model to the ICR strain and found the critical dose of caffeine to be 1.0 mg per mouse. Thus, we determined that a reasonable caffeine level was 1.0 mg per mouse per day by intraperitoneal injection. Wang et al. [15] reported no difference in serum concentrations between intraperitoneal and oral caffeine administration.

In the present study, the fertilization rate and blastocyst formation rate were decreased by caffeine administration, thus suggesting that caffeine negatively impacts oocytogenesis and embryonic development.

To date, most animal studies, conducted primarily in pregnant rats and mice, were designed to produce malformations; For example, caffeine is a teratogen that causes limb and palate malformations in rodents [16, 17, 18]. A report of positive effects of caffeine treatment found that fertilization capacity was increased by a short duration of exposure; however, 10 mM caffeine treatment did not affect the potential of fresh oocytes for fertilization and subsequent development [19]. Another study by Nash et al. [20] reported no fertility impact in the rat with a single intravenous injection of caffeine (25 mg/kg) on gestational day 6.

In the mouse, long‐term, continuous caffeine administration for at least two weeks decreased the number of retrieved oocytes; furthermore, using these oocytes for IVF resulted in a significantly decreased blastocyst formation rate; therefore, this suggests that caffeine affects oocyte quality.

Directly adding caffeine to the culture medium containing fertilized embryos resulted in a decreased blastocyst formation rate (in preparation for submission). A possible reason for this decrease is that the molecular structure of caffeine is very similar to adenosine. Therefore, it can be theorized that caffeine's primary mechanism of action is as an antagonist of adenosine [21]. Adenosine plays a role in the fundamental adenosine triphosphate (ATP)‐related energy metabolism and is necessary for RNA and DNA synthesis. Therefore, due to this similarity, caffeine can adversely impact embryo development.

The effect of testosterone and androstenedione on the early follicular stage in animals needs to essential and important action to produce oocytes [22]. Human studies have found a significant association between total testosterone and pregnancy rate [23]; furthermore, in vitro embryo development is dependent upon the amount of caffeine intake (in preparation for submission). These results strongly suggest that caffeine affects unknown regulatory mechanisms of testosterone, which in turn affects oocytogenesis. Sex hormone‐binding globulin (SHBG) levels were positively associated with increasing caffeine intake; thus, testosterone was decreased [24]. Caffeine was found to have a positive correlation with estrone and negative correlation with testosterone [25]. However, to date, we have not found any direct evidence of these correlations. Therefore, we must conduct further studies with animal models. Wang and Lau [15] reported no difference in serum caffeine concentration between intraperitoneal and oral caffeine administration in the rat. According to that study, these effects were similar following both routes of caffeine administration. The parallel pharmacokinetics for intraperitoneal and oral administration of caffeine were independently determined and related to the respective effects of caffeine on the reinforcement rate. Serum caffeine concentrations were similar across the session after the absorption phase for a given dose. Consequently, the effect remained in approximately the same range within a given dose, and no single dose possessed a full concentration–effect relationship for the two routes. However, it is necessary to develop comparable dosages for mice and humans.

When we added caffeine to the culture medium containing fertilized embryos and observed blastocyst formation, we found that, as the caffeine concentration increased, blastocyst formation, hatching, and number of hatched oocytes significantly decreased (data not shown). We currently have no understanding regarding the underlying mechanisms whereby caffeine also impacts embryo development. However, previous human studies have suggested that caffeine consumption can negatively influence fertility [26, 27, 28, 29]; these studies noted that a high caffeine intake prior to pregnancy appeared to be associated with an increased risk of spontaneous abortion, while surprisingly, low‐to‐moderate alcohol intake did not increase the risk. Specifically, high caffeine consumption may result in delayed conception [30]. An increase in coffee consumption was positively associated with the number of aborted pregnancies, while the number of good‐quality embryos decreased with high tea consumption [31].

If a woman regularly consumes a significant amount of coffee, her follicular fluid levels of total testosterone and androstenedione are decreased (in preparation for submission); as a result, the likelihood of pregnancy following ART will be decreased. Therefore, we must conduct more basic human studies.

Currently, the literature contains only a handful of reports addressing the relationship between caffeine and in vitro fertilization in rodents. Therefore, we attempted to confirm whether caffeine administration to mice would have a similar impact on ovarian function. We found similar effects for mice and humans.

In conclusion, caffeine administration prior to oocyte retrieval impacts oocytogenesis, and reduces the number of oocytes and the blastocyst formation rate in vitro. Thus, our findings suggest that caffeine affects embryo development as well as oocyte production in vivo. Further studies are needed in order to understand the mechanisms responsible for these effects.

Conflict of interest

We have no conflict of interest.