Lactoferrin treatment ameliorates endometriosis-associated subfertility in a rat model
Article information
Abstract
Objective
Endometriosis is a chronic inflammatory disorder that affects approximately 10% of reproductive-age women and is frequently associated with subfertility. This study evaluated the potential ameliorative effect of lactoferrin on endometriosis-associated subfertility in a rat model.
Methods
Rats were allocated into four groups: Sham, Sham+Lacto, Endo, and Endo+Lacto. In these group labels, “Endo” denotes endometriosis and “Lacto” denotes lactoferrin. Endometriosis was surgically induced in the Endo and Endo+Lacto groups, whereas rats in the Sham and Sham+Lacto groups underwent abdominal incision without uterine manipulation. Lactoferrin (300 mg/kg/day) was orally administered for 30 days to the Sham+Lacto and Endo+Lacto groups. After treatment, five females from each group were mated with mature males to assess fertility index parameters.
Results
Endo rats exhibited a significant increase in serum follicle-stimulating hormone (FSH) and a significant decrease in serum luteinizing hormone (LH) compared with Sham controls. In addition, marked histopathological alterations were observed in ovarian and eutopic uterine tissues. Maternal uterine and ovarian weights were significantly reduced in the Endo group, whereas fertility index parameters showed no significant differences among the experimental groups. Neonatal outcomes did not differ significantly between the Endo and Sham groups. In contrast, lactoferrin treatment significantly ameliorated gonadotropin imbalance, alleviated ovarian and eutopic endometrial histopathological alterations, and significantly increased neonatal weight and size in endometriotic rats, whereas neonatal length and pup number were not significantly changed.
Conclusion
Lactoferrin treatment ameliorated gonadotropin imbalance by improving serum FSH and LH levels, restoring ovarian and uterine tissue architecture, and enhancing fertility index parameters and neonatal outcomes. These findings suggest that lactoferrin may represent a promising non-hormonal approach for mitigating endometriosis-associated subfertility.
Introduction
The growth of endometrial-like tissue in ectopic locations is referred to as endometriosis, a chronic inflammatory condition [1]. Symptoms of endometriosis include debilitating pelvic pain, menstrual disorders, dysmenorrhea, and dyspareunia, all of which negatively affect quality of life [2]. It has been estimated that endometriosis affects approximately 10% of premenopausal women, and 30% to 50% of women with endometriosis experience infertility [3].
The diagnosis of endometriosis is often delayed because it relies on laparoscopy [4]. Current treatment options are limited to invasive surgery or medications that suppress estrogen production. These approaches are primarily directed toward symptom relief rather than definitive treatment and may have unfavorable effects on fertility [5].
Lactoferrin is a glycoprotein with multiple biological activities, including anti-inflammatory, antioxidant, antimicrobial, and anticancer effects. It also has iron-binding and immunomodulatory properties [6,7]. Lactoferrin is present at high concentrations in colostrum and is also found in exocrine secretions such as milk, saliva, tears, and gastrointestinal secretions. In addition, it is localized within the secondary granules of neutrophils [8,9].
Endometriosis-associated subfertility is likely multifactorial and may involve a disrupted pelvic microenvironment, inflammatory cytokines, altered redox status, and disturbed iron balance. These abnormalities may adversely affect fertilization and implantation and impair oocyte and embryo quality [10,11]. Given its iron-binding capacity and potent anti-inflammatory and antioxidant properties, lactoferrin may represent a promising non-hormonal therapeutic candidate for improving fertility outcomes in endometriosis [12,13].
Nevertheless, evidence regarding the impact of lactoferrin on endometriosis-associated subfertility remains limited. Therefore, the present study aimed to evaluate the potential ameliorative role of lactoferrin in experimentally induced endometriosis in rats by assessing reproductive performance and related histopathological and endocrine alterations.
Methods
1. Animals
Forty mature female Sprague-Dawley rats (8 to 10 weeks old; 170 to 200 g) were purchased from the Helwan Breeding Farm of the Egyptian Organization for Vaccine and Biological Preparations. The animals were housed in stainless steel cages in the animal house of the Zoology Department, Faculty of Science, Mansoura University, under standard conditions (22±2 °C; 12:12-hour light/dark cycle) with free access to standard chow and water. Approval to conduct the study was obtained from the Ethical Research Project Committee at Mansoura University (ACUC.SC.PhD.23.07.12).
2. Experimental groups
Daily vaginal smears were evaluated to determine the estrous cycle stage of each rat, and only rats in estrus were included in the study. Smears were assessed according to standard cytological criteria, specifically the predominance of cornified epithelial cells. After 1 week of acclimatization, rats were randomly divided into four groups, each comprising 10 animals: group I, Sham-operated rats; group II, Sham rats treated with lactoferrin (Sham+Lacto); group III, endometriosis control rats (Endo); and group IV, endometriotic rats treated with lactoferrin (Endo+Lacto). Randomization was performed using the envelope method, and investigators performing outcome assessment were blinded to group allocation.
3. Induction of endometriosis
According to the modified method of Vernon and Wilson [14], rats in the Endo and Endo+Lacto groups that were in estrus were used to establish the surgically induced endometriosis model. Thirty minutes after confirmation of estrus, the rats were anesthetized by intraperitoneal injection of a mixture of xylazine hydrochloride (5 mg/kg; Xylajet 20 mg/mL; ADWIA) and ketamine hydrochloride (75 mg/kg; Ketamax 50 mg/mL; Troikaa Pharmaceuticals Ltd.). The rats were placed on a heating pad to maintain body temperature at 37 °C. The ventral abdomen was aseptically prepared from the xiphoid cartilage to the pubic region and both lateral abdominal sides. A 5-cm midline incision was made in the ventral mesogastric region overlying the uterine horns. The left uterine horn was ligated, and an approximately 2-cm segment was resected. The resected tissue was opened longitudinally and immersed in sterile saline. It was then cut into four 5-mm² pieces, which were sutured to the inner abdominal wall using a simple interrupted pattern with 4-0 polyglactin 910 suture (Vicryl; Ethicon), with the endometrial surface facing the abdominal cavity. The abdominal layers were closed using a simple continuous pattern with 4-0 polyglactin 910 suture. The subcutaneous layer was closed with a subcutaneous suture pattern using 4-0 polyglactin 910 suture, and the skin was closed with a simple interrupted pattern using 2-0 silk suture. After surgery, rats were housed individually and received intramuscular ampicillin (15 mg/200 g) once daily for 3 consecutive postoperative days. Postoperative analgesia consisted of intramuscular meloxicam (4 mg/kg) administered for 3 days.
4. Confirmation of implants and treatment protocol
Four weeks postoperatively, a second abdominal surgery was performed to confirm the development of endometriotic implants. Successful model establishment was confirmed by the presence of visible, vascularized endometriotic implants at the second laparotomy. Five days after the second surgery, rats in the Endo+Lacto group received oral lactoferrin at a dose of 300 mg/kg/day for 30 consecutive days [15]. Lactoferrin was administered as a commercially available oral preparation (Pravotin Lactoferrin-100 sachets; HYGINT). The contents of each sachet were dissolved in distilled water to prepare a 75 mg/mL solution, such that 1 mL contained 75 mg of lactoferrin, and the solution was administered by oral gavage.
The lactoferrin dose (300 mg/kg/day) was selected on the basis of previous experimental studies indicating its safety and efficacy after oral administration in rats, together with preliminary pilot observations [16,17]. A 30-day treatment period was selected to encompass multiple estrous cycles and thereby allow adequate assessment of hormonal changes, reproductive tissue remodeling, and pregnancy outcomes in rats [18].
The Sham-operated and Sham+Lacto groups underwent the same anesthesia, abdominal incision, and suturing procedures, using the same suture patterns and materials described above, but without manipulation of the uterus. Five days after surgery, the Sham+Lacto group received oral lactoferrin at 300 mg/kg/day for 30 days.
5. Sample collection
Five rats from each group were euthanized after the treatment period by intraperitoneal injection of thiopental sodium (50 mg/kg; 0.5 g thiopental, EIPCO). Immediately before euthanasia, vaginal smears were obtained, and all animals were confirmed to be in estrus to minimize cycle-related variability in gonadotropin levels. Blood samples were collected by cardiac puncture, centrifuged at 3,000 rpm for 10 minutes to separate serum, and stored at −20 °C for subsequent analysis. Eutopic uterine tissues and ovaries were excised and fixed in 10% neutral formalin for histopathological evaluation.
1) Body weight
Body weights (g) of all rats were recorded at the beginning and end of the experimental period.
2) Determination of hormone levels
Serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels were measured using rat enzyme-linked immunosorbent assay kits supplied by CUSABIO (catalog numbers CSB-E06869r and CSB-E12654r, respectively), according to the manufacturer’s instructions.
3) Histological examinations
Eutopic uterine and ovarian tissues were fixed in 10% neutral formalin and then processed through dehydration, clearing, paraffin embedding, sectioning, and hematoxylin and eosin (H&E) staining. Sections (5 µm) were examined under a bright-field Olympus light microscope equipped with an AmScope MU1000 camera at 100× and 400× magnification [19].
6. Fertility index determination
Following the treatment period, five females from each group were housed with mature males at a ratio of one male to two females. The males were proven fertile (8 to 10 weeks old; 200 to 230 g), and cohabitation continued for 14 days. Males were randomly assigned across groups to minimize the influence of male fertility on reproductive outcomes. Mating was confirmed by the presence of sperm in vaginal smears. Pregnant females were then housed individually until delivery.
Three months after the initial endometriosis induction surgery, and after delivery, the dams underwent terminal dissection, and the maternal ovaries and uteri were excised. Corpora lutea, identified as raised opaque structures on the ovarian surface, were counted using a magnifying lens. In addition, implantation sites, recognized as placental scars in the uterus, were counted. Fertility potential (implantation index) was calculated as (number of implantation sites/number of corpora lutea)×100 [20,21]. Maternal ovarian and uterine weights were recorded. Litter number and pup size, length, and weight were also recorded. Selection of the five females per group for fertility testing was random, and outcome assessment was performed with investigators blinded to group allocation.
7. Reproductive performance indices
Reproductive performance indices were calculated as follows: mating index (%)=(number of females mated/number of females placed with males)×100; pregnancy index (%)=(number of females pregnant/number of females mated)×100; preimplantation loss (%)=[(number of corpora lutea−number of implantation sites)/number of corpora lutea]×100; and postimplantation loss (%)=[(number of implantation sites−number of neonates)/number of implantation sites]×100.
8. Statistical analysis
GraphPad Prism ver. 8 (GraphPad Software Inc.) was used for data analysis. Normality was assessed using the Shapiro-Wilk test. Results are presented as mean±standard error (SE). For neonatal outcomes, the litter was considered the experimental unit. Individual pup measurements, including pup weight, length, and size, were averaged within each litter, and each litter/dam contributed one data point to the analysis. Litter size, defined as the number of pups per litter, was recorded once for each dam and was also analyzed using the litter as the experimental unit. Group comparisons were performed using one-way analysis of variance followed by the Tukey post hoc test. A p-value <0.05 was considered statistically significant.
Results
1. Body-weight change
Figure 1 shows no significant differences in baseline or final body weight among the groups. Lactoferrin administration to Sham rats did not significantly affect body-weight change compared with Sham controls. In contrast, the Endo group showed a significantly smaller body-weight change than the Sham group (p≤0.01). Lactoferrin treatment did not significantly affect body-weight change in endometriotic rats compared with the Endo group.
Initial body weight (BW), final BW, and BW change in the different groups. Data are presented as mean±standard error of the mean (n=10 rats/group). a)p≤0.01 compared with the Sham group.
2. Follicle-stimulating hormone
As shown in Figure 2, lactoferrin administration to Sham rats did not significantly alter serum FSH levels compared with Sham controls. The Endo group exhibited a significant elevation in serum FSH levels compared with the Sham group (p≤0.001). Lactoferrin treatment significantly reduced serum FSH levels in endometriotic rats compared with the Endo group (p≤0.001).
Follicle-stimulating hormone (FSH) levels in the different groups. Data are presented as mean±standard error of the mean (n=5). a)p≤0.01; b)p≤0.001; c)Significant compared with the Sham group; d)Significant compared with the Endo group.
3. Luteinizing hormone
As shown in Figure 3, lactoferrin administration to Sham rats did not significantly alter serum LH levels compared with Sham controls. The Endo group showed a significant decrease in serum LH levels compared with the Sham group (p≤0.001). Lactoferrin treatment significantly increased serum LH levels in endometriotic rats compared with the Endo group (p≤0.001).
Luteinizing hormone (LH) levels in the different groups. Data are presented as mean±standard error of the mean (n=5). a)p≤0.001; b)Significant compared with the Sham group; c)Significant compared with the Endo group.
4. Histopathological findings
As shown in Figure 4, H&E-stained uterine sections from the Sham group demonstrated normal eutopic uterine architecture, including a simple columnar epithelium lining the uterine lumen and a cellular stroma containing tubular glands lined by regular columnar epithelial cells. The Sham+Lacto group displayed uterine histology comparable to that of the Sham group. In contrast, the Endo group showed histopathological alterations, including fewer endometrial glands within the stroma and epithelial degeneration with cytoplasmic vacuolation. Lactoferrin treatment improved uterine histology in endometriotic rats compared with the Endo group.
(A) Eutopic endometrial tissue in the Sham group showed normal histological architecture, with a regular simple columnar epithelium (E) lining the lumen (L) and a highly cellular stroma (S) containing tubular endometrial glands (G). (B) Eutopic endometrial tissue in the Sham+Lacto group showed a normal structure, with the uterine L lined by regular endometrial E and the endometrial S containing tubular endometrial G. (C) Eutopic endometrial tissue in the Endo group showed damaged endometrial E lining the uterine L, along with S containing fewer endometrial G. (D) Eutopic endometrial tissue in the Endo+Lacto group showed improved uterine histology compared with the Endo group, with endometrial E lining the L and S containing well-differentiated endometrial G (hematoxylin and eosin, ×100 and ×400).
As shown in Figure 5, ovarian sections from the Sham group demonstrated normal ovarian architecture, including normal stromal cells, follicles at various stages of maturation, and corpora lutea. The Sham+Lacto group showed ovarian histology comparable to that of the Sham group. In contrast, the Endo group exhibited histopathological changes, including reduced numbers of ovarian follicles across developmental stages and vacuolated stromal cells. Lactoferrin treatment ameliorated these ovarian histopathological alterations compared with the Endo group.
(A) Ovarian tissue in the Sham group showed normal histological architecture, including primary follicles (PF), secondary follicles (SF), Graafian follicles (GF), well-developed corpora lutea (CL), and blood vessels (BV). (B) Ovarian tissue in the Sham+Lacto group showed normal structure, with several ovarian follicles, including PF, SF, GF, and CL. (C) Ovarian tissue in the Endo group showed only PF and CL, along with some vacuolated stromal cells (arrowheads). (D) Ovarian tissue in the Endo+Lacto group showed improved ovarian histology compared with the Endo group, with several ovarian follicles, including PF, SF, and CL (hematoxylin and eosin, ×100 and ×400).
5. Fertility index findings
1) Maternal organ weights
As shown in Figure 6, lactoferrin administration to Sham rats did not significantly affect maternal uterine or ovarian weights compared with Sham controls. Compared with the Sham group, the Endo group exhibited significantly lower maternal uterine and ovarian weights (p≤0.05). Lactoferrin treatment produced a nonsignificant increase in maternal uterine and ovarian weights compared with the Endo group.
Maternal (A) ovarian weight and (B) uterine weight in the different groups. Data are presented as mean±standard error of the mean (n=5 dams/group). a)p≤0.05 compared with the Sham group.
2) Fertility potential
As shown in Figure 7, lactoferrin administration to Sham rats did not significantly affect fertility potential compared with Sham controls. The Endo group showed no significant reduction in fertility potential compared with the Sham group (p≥0.05). Lactoferrin treatment produced a trend toward increased fertility potential compared with the Endo group; however, this change did not reach statistical significance.
Fertility potential in the different groups. Data are presented as mean±standard error of the mean (n=5 dams/group). No statistically significant differences were detected among the groups.
3) Neonatal outcomes
As shown in Figure 8, lactoferrin administration to Sham rats did not significantly affect neonatal weight, length, size, or pup number compared with Sham controls. Compared with the Sham group, the Endo group showed no significant differences in these neonatal outcomes (p≥0.05). Compared with the Endo group, lactoferrin treatment significantly increased neonatal weight and size in endometriotic rats (p≤0.01), whereas increases in neonatal length and pup number were not statistically significant. These data are presented in Table 1.
Neonatal outcomes: (A) mean pup weight per litter, (B) mean pup length per litter, (C) mean pup size per litter, and (D) number of pups per litter in the different groups. Individual pup measurements were averaged within each litter, and each litter was considered one experimental unit. Data are presented as mean±standard error of the mean (n=5 L/group; 1 L per dam). a)p≤0.01 compared with the Endo group.
Reproductive performance indices in the studied groups
Discussion
Endometriosis is a chronic, inflammatory, hormone-dependent condition that negatively affects fertility [22]. Lactoferrin is a multifunctional glycoprotein with anti-inflammatory, antioxidant, and immunomodulatory properties [23]. Accordingly, the present study evaluated the potential ameliorative effect of lactoferrin on endometriosis-associated subfertility in an experimentally induced rat model of endometriosis.
In the present study, serum FSH levels were significantly elevated, whereas LH levels were significantly decreased, in endometriosis rats compared with Sham rats. These hormonal disturbances are consistent with previous reports suggesting that disruption of the hypothalamic-pituitary-ovarian axis may result from the inflammation and hormonal imbalance associated with endometriosis, leading to ovulatory and endometrial abnormalities [10,24].
The histopathological findings of the present study support these hormonal changes. Ovarian tissue from the Endo group showed degenerative alterations in the ovarian cortex, together with reduced numbers of developing follicles, compared with the Sham group. These findings are consistent with previous reports indicating that peritoneal oxidative stress and inflammation may disrupt folliculogenesis and ovulation, thereby reducing ovarian reserve [25,26]. Furthermore, the impaired oocyte quality associated with endometriosis may reduce embryo quality and implantation potential [27].
In addition, the present study demonstrated histopathological alterations in the eutopic endometrium of Endo rats that may adversely affect implantation. These findings are consistent with previous reports indicating that the hormonal imbalance, altered redox status, and inflammatory mediators associated with endometriosis may induce structural and functional changes in the eutopic endometrium, thereby impairing implantation or fetal growth [27,28]. Moreover, progesterone resistance in endometriosis has been associated with luteal phase defects that may negatively affect endometrial receptivity [29].
The present results showed that maternal reproductive organ weights were significantly reduced in Endo rats compared with Sham rats, whereas fertility-related parameters showed only a nonsignificant downward trend. Although implantation efficiency did not differ significantly among groups, the improved neonatal outcomes observed in the Endo+Lacto group suggest that lactoferrin may improve the endometrial and fetal developmental environment [30,31].
These findings are consistent with reports indicating that peritoneal inflammation, redox imbalance, altered hormonal secretion, immune dysfunction, and pelvic anatomical changes associated with endometriosis may adversely affect ovulation, oocyte transport, sperm function, embryo development, and implantation potential [10,11,32]. Notably, lactoferrin treatment ameliorated gonadotropin imbalance, alleviated ovarian and eutopic endometrial histopathological alterations, and improved several fertility-related parameters in Endo rats. These ameliorative effects of lactoferrin may be attributable, at least in part, to its anti-inflammatory, antioxidant, and immunomodulatory properties, consistent with previous reports [33-35].
Although the present study did not directly assess inflammatory cytokines, oxidative stress biomarkers, or immune cell infiltration, the observed improvement in gonadotropin balance and reproductive tissue histology indirectly supports the proposed anti-inflammatory and antioxidant effects of lactoferrin, consistent with previous literature [6,36].
Lactoferrin is a glycoprotein with minimal reported toxicity [37]. It has also been evaluated under the U.S. Food and Drug Administration Generally Recognized as Safe notification program for use as a food ingredient [38]. In addition, it may represent a potential non-hormonal therapeutic candidate for endometriosis compared with currently used medical therapies, such as combined oral contraceptives, progestins, and gonadotropin-releasing hormone agonists/antagonists, which rely primarily on hormonal suppression. Although such therapies may provide symptom relief, they are associated with hypoestrogenic adverse effects and are generally unsuitable for women attempting conception [11,39].
Moreover, given lactoferrin’s favorable safety profile in clinical and nutritional research, dose extrapolation based on body surface area suggests that the selected dose falls within a biologically relevant and safe range for human oral supplementation [13,40].
The present study has several limitations. First, the sample size used for fertility evaluation was relatively small, and direct evaluation of ovarian reserve markers, such as anti-Müllerian hormone (AMH) levels and follicle count, as well as inflammatory and oxidative stress biomarkers, was not performed. Second, histological evaluation was mainly qualitative, and quantitative follicle counts and endometrial morphometry were not included. Therefore, although the present results support improvements in selected reproductive and neonatal outcomes, direct evaluation of AMH and follicle count is required before definitive conclusions can be drawn regarding changes in ovarian reserve. To better elucidate the mechanistic pathways underlying lactoferrin-mediated amelioration, future studies with larger cohorts and direct assessment of inflammatory cytokines, oxidative stress markers, and immune cell infiltration in ectopic and eutopic tissues are warranted.
In conclusion, although lactoferrin did not significantly restore implantation efficiency, it improved postimplantation pregnancy-related outcomes, as reflected by increased neonatal weight and size. Overall, the present findings suggest that lactoferrin mitigates several endocrine and histopathological alterations associated with endometriosis in a rat model, including improved gonadotropin balance and amelioration of ovarian and eutopic endometrial histology. These findings support lactoferrin as a potential non-hormonal therapeutic candidate for improving selected pregnancy-related outcomes under endometriotic conditions, possibly through anti-inflammatory and antioxidant actions. However, further experimental and clinical studies incorporating comprehensive fertility assessment, ovarian reserve markers (e.g., AMH and quantitative follicle counts), and direct measurement of inflammatory and oxidative stress biomarkers are required to confirm these findings, clarify the underlying mechanisms, and strengthen their translational relevance.
Notes
Conflict of interest
No potential conflict of interest relevant to this article was reported.
Author contributions
Conceptualization: MMAB, MA, HAEG. Methodology: MMAB, MA, HAEG. Formal analysis: MMAB, MA, HAEG. Data curation: MMAB, MEA, MA, MAE. Visualization: MMAB, MEA, MA, MAE. Software: MMAB, MEA, MA. Validation: MMAB, MEA, MA. Investigation: MMAB, MEA, MA. Supervision: MMAB, MA, HAEG. Writing-original draft: MMAB, MA. Writing-review & editing: MMAB, MA. Approval of final manuscript: MMAB, MEA, MA, MAE, HAEG.