Role of L-carnitine in female infertility

Though a minute functional difference between LC and ALC has been reported, both are used in reproductive biology research to improve mitochondrial functions to treat infertility [5, 8, 9]. ALC is used most often associated with the improvement of antioxidant/anti-aging effects, while LC is commonly used to improve the body’s ability to oxidize fat cells which assists in the production of energy and burning fat [27]. During oocyte development, it has been reported that the cumulus-oocyte complex (COC) plays an essential role in lipid metabolism and energy production. Thus, in oocytes, maintenance of proper lipid oxidation with/without a minimum production of free radicals is crucial to preserve its quality [30]. Though it is well documented that both forms of carnitine possess antioxidant properties [10], it has been suggested in some reports that ALC is more effective in combating ROS-induced oxidative damage compared to LC [15]. Thus, studies focusing on antioxidant defense-mediated improvement of female fertility preferentially utilize ALC, whereas, studies demonstrating improved lipid metabolism-induced enhancement of oocyte quality, and thereby fertility, tend to use LC. Other acylcarnitines are also used in reproductive biology research, but to a lesser extent [35]. In 2013, Varnagy et al. reported the profiling of several acylcarnitines in the serum and follicular fluids of women undergoing IVF. They provided suggestive evidence that in IVF patients with better reproductive potential (higher number of oocytes and/or viable embryos), the LC/acylcarnitine pathway appears to be upregulated while the endogenous carnitine pool is diminished, which consequently improves oocyte quality [35]. In the following sections, we will provide evidence of LC and/or ALC supplementation and their role in female fertility.

Most of the human experiments were carried out using LC as a supplement to alleviate/combat the problem of female infertility [11, 18, 36] (Table 1). Several studies found that both LC and ALC supplementation improves disorders such as polycystic ovary syndrome [12], endometriosis [19] and amenorrhea [17]. Carnitines are reported to increase gonadotropins and sex hormone levels as well as improve oocyte health [17]. However, the effects of LC on endometriosis are still a matter of debate [19, 29, 37, 38]. The possible mechanistic pathways through which LC could exert its effects on endometriosis will be discussed in a subsequent section.

Polycystic ovary syndrome (PCOS) is a common female reproductive endocrinopathy. The main pathophysiological mechanisms underlying this syndrome include obesity, insulin resistance and hyperinsulinemia. Samimi and colleagues found that LC supplementation (250 mg per day orally for 12 weeks) lead to significant reduction in body weight, BMI, waist and hip circumference respectively as well as improved glycemic control in women with PCOS (mean age 24.8 ± 5.5 years) [12]. This study indicated that LC supplementation improves PCOS by decreasing blood glucose levels and opposing insulin resistance [12], which could perhaps be attributed to LC-induced increase in beta-oxidation of fatty acids and basal metabolic rates [39].

Women with PCOS also have an imbalance between male and female hormones as their ovaries tend to produce androgens in excessive amounts. One study suggested that hyperandrogenism and/or insulin resistance in the non-obese women with PCOS may be associated with decreased total serum LC levels [36]. Fenkci’s group had measured the serum total LC levels in non-obese women with PCOS (n = 27; aged between 16 to 37 years) in comparison to that of healthy adult women (n = 30). They demonstrated these PCOS patients have significantly lower total LC (40.5 ± 5.7 μmol/L; in control: 91.1 ± 15.2 μmol/L), but higher levels of dehydroepiandrosterone (DHEA), testosterone, luteinizing hormone (LH), low-density lipoproteins (LDL) and fasting insulin compared to healthy women [36].

Another common feature of PCOS is chronic anovulation. The standard approach to treat women with anovulatory infertility, secondary to PCOS, is to administer clomiphene citrate to induce ovulation. However, some women do not ovulate despite receiving increasing doses of clomiphene citrate, and are therefore considered to be clomiphene citrate-resistant. In a single center, double blinded, randomized controlled clinical trial, Ismail et al. studied the effect of LC supplementation on clomiphene-resistant women with PCOS. All subjects received 250 mg clomiphene citrate daily from day-3 until day-7 of the cycle. In addition, women in the treatment arm (n = 85) were supplemented daily with 3 g of LC orally (day-3 until day of first positive pregnancy test), while those in the control arm (n = 85) received a placebo. Follicular maturation was monitored using transvaginal ultrasonography on all patients on day-7 and day-9, and subsequently adjusted according to individual response [11].

Ismail’s group found that LC supplementation along with clomiphene citrate treatment improved both ovulation (64.4% vs.17.4%; < 0.0001) and pregnancy (51.5% vs. 5.8%; p < 0.0001) rates in clomiphene-resistant women with PCOS. LC supplementation seemed to improve the number and rate at which the stimulated follicles developed (to a diameter of ≥17 mm for induction of ovulation), increase beta-oxidation and oocyte maturation as well as increase serum levels of both estradiol (E₂) and progesterone. LC supplementation not only improved reproductive health, but also enhanced the patients’ lipid profile and body mass index (BMI) [11].

A typical alternative treatment to induce ovulation in clomiphene citrate-resistant PCOS patients is gonadotropin therapy. However, some women with PCOS fail to respond to both these treatments. Latifian et al. studied the effects of LC on 50 infertile women with PCOS who were both clomiphene- and gonadotropin-resistant. These women (mean age 27.98 ± 4.61 years) showed improvement following LC supplementation, which was given 2 g orally every 12 h (from the third day of the ovarian stimulation cycle onwards until the HCG injection). The LC-treated women in this study experienced the growth of dominant follicles (64%, n = 32) and displayed a positive pregnancy test (20%, n = 10). Moreover, LC was observed to increase the mean endometrium thickness as well as to inculcate positive changes in the left ovarian follicle size [18].

Not only does LC supplementation improve reproductive parameters in PCOS patients, but it can also confer positive effects on other health parameters in these women. This was demonstrated in a recent study whereby oral LC supplementation (250 mg for 12 weeks) among patients with PCOS improved total antioxidant capacity (TAC), decreased lipid peroxidation and enhanced general and mental health parameters [40].

Functional hypothalamic amenorrhea (FHA) is a form of hypogonadotropic hypogonadism that results from an aberration in the pulsatile release of hypothalamic gonadotropin-releasing hormone (GnRH), which causes decreased gonadotropin release leading to reduced estradiol production in the ovary [41]. ALC administration (1 g/day orally for 16 weeks) in patients suffering from FHA (n = 24, age range 21 to 32 years) was found to increase LH levels by counteracting certain neuroendocrine pathways that have an inhibitory effect on the reproductive axis [17]. Genazzani and co-workers proposed that ALC acts through the opioidergic pathway and alters protein/hormone functions by acetylating -OH groups in amino acids like serine, threonine or tyrosine, thereby improving their functions. They concluded that ALC has positive therapeutic effects on the reproductive axis in hypogonadotropic women with FHA and improves the condition by acting through the HPG axis [17].

Numerous research studies have been carried out in animal models (murine and farm animals) to investigate how LC and ALC improve female reproductive functions [16, 19, 21, 42‐44] (Table 1). Carnitines have been showed to have beneficial impacts in most of the studies involving PCOS, endocrine disorders and other cases of infertility. However, as alluded earlier, carnitines appear to possibly exert mixed effects on endometriosis [19, 29, 37, 38].

Despite the aforementioned beneficial effects of LC and ALC treatment on female fertility, Dionyssopoulou et al. found that carnitine supplementation modified the production of various cytokines, such as interferon-γ, tumor necrosis factor-α, interleukins-2, − 4, − 6, vascular endothelial growth factor, granulocyte-macrophage colony stimulating factor, and insulin-like growth factor-1 in serum and peritoneal fluid as tested using the immunofluorescence or ELISA technique [19]. Furthermore, carnitine treatment altered the percentage of immune cells (macrophages, T cells, CD4⁺ and CD8⁺ cells) in peritoneal exudates and uterine cells. Immune modifications such as these as well as the presence of cytokines and growth factors in the peritoneal cavity may contribute to the development of endometriosis. The potential role of prostaglandins (PG), including that of PGE₁ and PGE₂, are directly associated with inflammation and could affect the function of immune cells. It was found that while inhibition of PGE₁ further increased the levels of certain cytokines in serum and peritoneal fluid [45], inhibition of PGE₂ reversed these values toward that of control [46]. Thus in presence of LC, it was suggested that PGE₂ plays a role in the mechanistic pathway leading to endometriosis, while PGE₁ may potentially be involved in suppressing the production of immune cells and inflammatory response [29].

In 1992, Krsmanovic et al. examined the impact of ALC (50 mg/kg/day for a month) on the HPG axis in female rats and found that serum LH and prolactin levels were increased during the proestrous and estrous phases, while serum E₂ levels were increased during the estrous phase of the cycle. They have also noted an increase in uterine weight during both the proestrous and estrous phases of ALC-treated animals. Release of basal GnRH was found to be increased during the proestrous and diestrous I in hypothalamic slices of ALC-treated rats, which could mean that ALC stimulates the secretory activity of GnRH-producing hypothalamic neurons. In contrast, no alteration in pituitary functions was detected following ALC treatment in vitro. From these results, it was concluded that ALC may act through the HPG axis and cause gonadotropin release and this action is dependent on the cycle stage [16].

Virmani et al. reported in 2015 that treatment of mice with both LC (0.4 mg/mouse) and ALC (0.12 mg/mouse) resulted in increased number of mature oocytes and less degradation of oocytes. They concluded that LC and ALC co-treatment showed better response in maintaining the quality and quantity of oocytes in mice. In females, an age-related decline in the number and quality of follicles and oocytes have already been documented [47]. Aging and ROS affects many biochemical pathways of developing oocytes that may produce a deleterious impact on oocyte health and in this aspect, LC supplementation has showed some beneficial roles. Christiana’s group investigated another potential effect of LC through their study in which young BALB/c mice were given LC supplementation (2.5 mg/day/mouse for 7 days), superovulated and then mated with healthy males. They showed that 7 day-LC administration alters the lipid body content in pre-implantation embryos and did not result in live births in these females. This is because lipid body content is an important contributory factor towards healthy oocyte and embryo development and therefore its alteration may lead to infertility [37]. Conversely, a recent report by Virmani et al. (2017) showed that supplementation of carnitines (LC 0.4 mg/mouse + ALC 0.12 mg/mouse) to 8-week-old female CD1 mice not only improved oocyte formation but also increased the number of live birth in superovulated mice [48].

Fakhrildin and Flayyih reported an improvement in weight of the reproductive organs (ovaries, uterine horns, vagina) and endometrial thickness, litter sizes as well as serum LH, FSH and E2 levels in pregnant mice (Swiss albino strain females aged between 12 to 14 weeks) given 0.5 and 1.0 mg/kg LC compared to the control group. As the parameters measured did not differ significantly between the two LC doses used, the study concluded that a low dose of LC was sufficient to render positive effects on pregnancy and offspring outcomes in pregnant mice [42].

Canbolat and co-worker’s study investigated the antioxidant property of LC (100 and 500 mg/kg/day) on oxidative stress parameters (nitric oxide (NO), malondialdehyde, total antioxidant status, total oxidative stress (OS), and OS index in the kidney, liver and heart, as well as sera of bilateral oophorectomized rats. They have reported LC exhibits antioxidant effects in different tissues with less NO production, lipid peroxidation and OS index when supplemented with 500 mg/kg/day LC intraperitoneally for 14 consecutive days in surgically menopausal rats [49].

Several studies have also looked into the effects of carnitine supplementation on improving the rate of ovulation and fertilization [20], egg quality [44, 50], number of litters born [20, 51] etc., of farm animals such as pigs, chickens and cows.

Samland et al. examined the effect of dietary supplementation of LC (200 ppm) on the ovulation and fertilization rate of gilts (young female adult breeding pigs that have not yet farrowed). After two weeks, the gilts fed with added carnitine were found to have increased rate of ovulation, but decreased fertilization rate of the recovered embryos. However, the report did not offer an explanation as to the possible cause of decreased fertilization rates despite increased ovulation rates in this group of gilts [20].

Zhai and co-workers examined the effect of dietary supplementation of LC (125 ppm) on the reproductive traits of male and female White Leghorn chicken. They reported that the yolk of fresh eggs retrieved from LC-hens that were inseminated with semen from roosters consuming either the control or the LC-diet contained higher concentrations of LC but decreased hatchling yolk sac weights and yolk sac lipid content compared to that of control hens. This suggests that LC encourages fat utilization in the developing embryos [43].

In another study using gestating sows (mature female pigs that have farrowed at least once before the current gestation), Musser et al. demonstrated that a 50 ppm LC supplementation in gestation sow diet improved muscle fiber development by increasing the leanness and muscling of the fetal piglet. These changes could perhaps improve postnatal growth in the piglets [52].

In another study on the performance and egg quality parameters of Brown egg-type laying hens, Corduk and Sarica reported that supplementation of 500 mg LC per kg of low energy diets containing either sunflower or palm oil increases the eggshell breaking strength. Moreover, the addition of LC into normal diet containing palm oil increased the albumen index (calculated from the albumen height, length and weight) of the eggs [50]. Greater eggshell breaking strength and a higher albumen index indicates higher egg quality.

Pirestani and colleagues evaluated the effect of LC supplementation (50 g/day/cow) for 40 days on milk somatic cell count (SCC) (as a measure of the milk quality) and several reproductive indices in Holstein dairy cattle. They reported a significant reduction in milk SCC (indicating less mastitis or enhanced udder immunity and therefore better quality of milk) and improvement in the reproductive indices. In addition, the study found that a combination of LC and a vitamin-like essential macronutrient, choline (60 g/day/cow) further reduced milk SCC and improved the reproductive indices [21].

In another study on German Holstein dairy cows, Scholz’s group showed that supplementation with 2 g of LC/cow (contained in 10 g of a rumen-protected product) for three months improved metabolic health during the critical periods of transition and high lactation and showed a trend of improving milk production during the initial 2-months after calving. Furthermore, LC-supplemented cows had a lower insemination index and increased conception rate compared to controls [53].

Following reports on the antioxidant properties of LC and ALC and its beneficial effects on female fertility, carnitines have been used in in vitro studies focusing on the improvement of oocyte health and maturation, embryo development as well as in assisted reproduction [54, 55] (Table 2). In the last two decades, a good number of studies have been published that showcase the impact of carnitine supplementation on female fertility. Of late, it has been used to minimize ROS-mediated delayed embryonic development in culture medium, high DNA fragmentation and development of morphologically abnormal blastocysts after prolonged culture [6, 23, 56].

During in vitro fertilization (IVF) procedures, embryo fragmentation due to apoptosis is a common occurrence that is well documented. Nonetheless, supplementation of culture medium with LC may confer protection to the developing immature cells. Pillich et al. have shown that ALC supplementation (0.3, 0.6 and 1.2 mM for 5 h and 24 h) in mouse fibroblast culture media stabilizes the mitochondrial membrane, increases energy supply to the organelle and protects the developing cell from apoptosis through the mitochondrial pathway [57]. In another laboratory, Abdelrazik and colleagues looked into the optimal dose of LC that is required for blastocyst development of mouse embryos. They have showed that 0.3 and 0.6 mg/mL of LC possesses anti-apoptotic effects as well as increased the rate of blastocyst development [33].

A marked increase in TNF-α concentration in granulosa cell cultures of women with endometriosis has been demonstrated [58‐61]. Studies have also shown that increased levels of TNF-α restrict inner cell mass and trophectoderm proliferation in mouse blastocyst. At a concentration of 50 ng/mL, TNF-α was found to affect protein synthesis in mouse embryos in both morula and blastocyst stages [62, 63]. However, LC at the doses of 0.3 and 0.6 mg/mL were able to neutralize the anti-proliferative effects on TNF-α. LC supplementation in embryo culture medium also decreased DNA damage during development [6].

These observations were reaffirmed by Zare and co-workers who used the same doses (0.3 and 0.6 mg/mL) of LC supplementation during in vitro maturation of immature BCB+ (Brilliant Cresyl Blue positive) oocytes. LC-treated oocytes demonstrated an improved pre-implantation developmental competence (quality) after IVF, which is probably due to LC-induced improvement in the cytoplasmic and nuclear maturation of immature oocytes. LC at the doses used also exhibited an antioxidative effect during embryo development by reducing ROS levels in the maturation medium [64].

In another study, Mansour et al. used the same dose (0.6 mg/mL) of LC to demonstrate the protective effects of LC on oocytes and embryos against the toxic effects of peritoneal fluid in women with endometriosis. They have showed that peritoneal fluid of patients with endometriosis which was supplemented with LC had decreased apoptosis levels in the embryos and improved oocyte microtubular and chromosomal structure [38]. Bareh has also reported that LC-induced improvements in cytoskeleton could lead to reduced aneuploidy rates in an animal model [65].

In 2013, Phongmitr et al. added LC (0.3, 0.6 and 1.2 mg/mL) to culture media containing swamp buffalo oocytes and noted an improved nuclear maturation rate with a maximum number of metaphase II (MII) oocytes in the 0.3 mg/mL LC-supplemented group [66]. Moawad’s group later showed that LC supplementation (0.6 mg/ml or 3.72 mM) during vitrification/warming and in vitro maturation of germinal vesicle stage-oocytes increased the proportions of oocytes with normal MII spindles. They also reported that LC supplementation causes increased oxidative activity of mitochondria in the resultant MII oocytes without increasing the ATP levels in MII oocytes [55].

Ghanem and co-workers reported similar observations of increased antioxidant activity and improved blastocyst quality by way of higher embryo development rate and fewer number of apoptotic cells after supplementing LC (1.5 mM, equivalent to 0.3 mg/mL) in culture media of bovine blastocyst during the pre-implantation stage [67]. Similar studies conducted earlier have also shown higher counts of total cells [68, 69] and lower apoptotic cells [69] in bovine embryos cultured in 0.3 mg/mL L-carnitine.

Manzano’s group investigated the role of L-carnitine supplementation in the maturation of bovine oocytes and pre-implantation development of embryos. They reported improved developmental potential up to the blastocyst stage with higher blastocyst formation rate and increased total cell count and activity when the oocytes were matured in culture media added with 0.1–0.5 mg/mL LC. In a subsequent experiment, supplementation of the resulting zygotes with the same dose of LC in modified synthetic oviductal fluid medium displayed a higher blastocyst total cell count, but similar blastocyst formation rates as that of control [70].

Khanmohammadi and colleagues evaluated the effect of LC (0.5 mg/mL) supplementation on several indicators of embryo development and blastocyst quality such as thickness of zona pellucida, hatching rate and number of blastocyst cells. At a concentration of 0.5 mg/mL, LC exerted antioxidant properties, along with an enhancement of mitochondrial lipid metabolism to increase blastocyst cell number, blastocyst expansion and zona pellucida thinning, all of which promotes blastocyst quality leading to successful hatching and implantation. Conversely, LC at high concentrations (4 mg/ml) had a toxic effect on in vitro embryo development and blastocyst quality [71].

LC has been reported to maintain cellular energy [34], reduce oxidative stress [72] and minimize cell death by apoptosis [33], which are necessary for proper oocyte growth and maturation of blastocyst. As discussed earlier, the cumulus-oocyte complex (COC) and its lipid metabolism are one of the prime regulators of oocyte maturation [73]. LC helps in the metabolism of COC lipids by transferring fatty acids into the mitochondria and by facilitating β-oxidation [30]. LC is taken up by the tissues via the electrogenic force of the voltage-gated Na⁺-channels. It uses the Na⁺-driven LC / organic cation transporter-2 (OCTN-2) for its transport into the oocytes [74].

In the oocytes, LC gets converted to ALC by carnitine palmitoyltransferase-I (CPT-I) in the outer mitochondrial membrane and CPT-II helps in the regeneration of carnitine from acyl-carnitine after the translocation of long chain fatty acids into the mitochondrial matrix [31]. Within the oocyte, LC plays a significant role in ER, mitochondria as well as in the ooplasm. In the mitochondria, LC is converted to ALC, and balances the acetyl CoA/CoA ratio to maintain glucose metabolism through the TCA cycle, yielding a higher energy production [34]. LC helps in minimizing the concentration of pyruvate which prevents entry into the TCA cycle to curtail energy production (Fig. 2).

In the mitochondria, LC also scavenges ROS through its antioxidant property. It transports palmitate and other long chain fatty acids to the mitochondria to facilitate their utilization through β-oxidation [5]. In the ER, it decreases the concentration of palmitate by transferring it to the mitochondria or by eliminating it from which it may cause lipotoxicity of the oocytes through oxidative stress [34]. It is reported that higher ROS levels decrease maturation by affecting the COC, and also decreases embryo development and blastocyst fragmentation [75]. LC also promotes cellular proliferation and decreases apoptosis by inhibiting TNF-α and other anti-proliferative agents [33]. In endometriosis, LC has been claimed to possess dual effects through the uterine secretion of prostaglandins. LC treatment has been shown to increase the production of both PGE₁ and PGE_2. It was suggested that PGE₁ confers protection against endometriosis by attenuating the secretion of TNF-α, IFN-γ and interleukins (IL-2, IL-4 and IL-6), while PGE₂ induces cytokine release and thus endometriosis through its apoptotic and inflammatory functions [19, 46]. However, further robust studies are required to corroborate the findings regarding the role of LC on endometriosis.

Thus, in summary, free LC or ALC serves three important functions in oocytes through its direct action: first, it increases energy production by transferring palmitate into mitochondria and maintaining acetyl CoA/CoA ratio; secondly, it reduces oxidative stress and lipotoxicity by scavenging free radicals and removing excess palmitate from the ER, and finally it promotes oocyte growth and maturation by decreasing the rate of apoptosis.

LC and ALC have also been reported to affect the HPG axis to promote reproductive hormone secretion [16, 17, 76]. It has been known that among the neural centers, LC’s concentration is highest is in the hypothalamus [77]. In the hypothalamus, LC has been reported to decrease neuronal cell death and damage associated with aging [78] by its cholinomimetic activity [79]. It is reported that LC increases GnRH secretion from the hypothalamus by acting on the HPG axis [16, 17] and causing K⁺-induced depolarization in hypothalamic neuronal cells to increase its secretory activity [80, 81]. They have shown that treatment with ALC increases serum levels of other reproductive hormones, like estradiol, progesterone, LH and decreases prolactin in the usual fashion with different phases of estrous in animal [16, 17]. Through its indirect endocrine effect, it prevents PCOS, amenorrhea and other problems related to the female reproductive cycle.

Costa and Stevenson (1984) with the administration of equine chorionic gonadotropin (eCG) and pregnant mare’s serum gonadotropin (PMSG) showed that these components increases ovarian concentration of free LC and ALC, which shows the interaction between the direct and indirect mechanisms through cellular and hormonal regulations [82].