{"paper_id":"e68effe8-f62a-4120-8be5-39c813c45c0a","body_text":"© 2026 THE KOREAN SOCIETY FOR REPRODUCTIVE MEDICINE www.eCERM.org 1\nThis is an Open Access article distributed under the terms of the Creative Commons Attribu-\ntion Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0/) which permits \nunrestricted non-commercial use, distribution, and reproduction in any medium, provided the \noriginal work is properly cited.\nLactoferrin treatment ameliorates endometriosis-\nassociated subfertility in a rat model\nMona M. Abdel Baky\n1\n, Maggie E. Amer\n1\n, Marwa Abass\n2\n, Mohammed A. Elmetwally\n3\n, Heba A. El-Ghaweet\n1\n1\nZoology Department, Faculty of Science, Mansoura University, Mansoura; \n2\nDepartment of Surgery, Anesthesiology, and Radiology, Faculty of \nVeterinary Medicine, Mansoura University, Mansoura; \n3\nDepartment of Theriogenology, Faculty of Veterinary Medicine, Mansoura University, Mansoura, \nEgypt\nORIGINAL ARTICLE\nhttps://doi.org/10.5653/cerm.2026.09166\npISSN 2233-8233 · eISSN 2233-8241\nClin Exp Reprod Med [Epub ahead of print]\nReceived: January 2, 2026 ∙ Revised: February 19, 2026 ∙ Accepted: March 19, 2026\nCorresponding author: Marwa Abass\nDepartment of Surgery, Anesthesiology, and Radiology, Faculty of Veterinary \nMedicine, Mansoura University, Mansoura 35516, Egypt\nTel: +20-1015217659 Fax: +20-1015217659 E-mail: marwa_mossa@mans.edu.eg \nObjective: Endometriosis is a chronic inflammatory disorder that affects approximately 10% of reproductive-age women and is frequently \nassociated with subfertility. This study evaluated the potential ameliorative effect of lactoferrin on endometriosis-associated subfertility in a \nrat model.\nMethods: Rats were allocated into four groups: Sham, Sham+Lacto, Endo, and Endo+Lacto. In these group labels, “Endo” denotes endome-\ntriosis and “Lacto” denotes lactoferrin. Endometriosis was surgically induced in the Endo and Endo+Lacto groups, whereas rats in the Sham \nand Sham+Lacto groups underwent abdominal incision without uterine manipulation. Lactoferrin (300 mg/kg/day) was orally administered \nfor 30 days to the Sham+Lacto and Endo+Lacto groups. After treatment, five females from each group were mated with mature males to as-\nsess fertility index parameters.\nResults: Endo rats exhibited a significant increase in serum follicle-stimulating hormone (FSH) and a significant decrease in serum luteinizing \nhormone (LH) compared with Sham controls. In addition, marked histopathological alterations were observed in ovarian and eutopic uterine \ntissues. Maternal uterine and ovarian weights were significantly reduced in the Endo group, whereas fertility index parameters showed no \nsignificant differences among the experimental groups. Neonatal outcomes did not differ significantly between the Endo and Sham groups. \nIn contrast, lactoferrin treatment significantly ameliorated gonadotropin imbalance, alleviated ovarian and eutopic endometrial histopatho-\nlogical alterations, and significantly increased neonatal weight and size in endometriotic rats, whereas neonatal length and pup number \nwere not significantly changed.\nConclusion: Lactoferrin treatment ameliorated gonadotropin imbalance by improving serum FSH and LH levels, restoring ovarian and uter-\nine tissue architecture, and enhancing fertility index parameters and neonatal outcomes. These findings suggest that lactoferrin may repre-\nsent a promising non-hormonal approach for mitigating endometriosis-associated subfertility.\nKeywords: Endometriosis; Inflammation; Lactoferrin; Subfertility\nIntroduction\nThe growth of endometrial-like tissue in ectopic locations is re-\nferred to as endometriosis, a chronic inflammatory condition [ 1]. \nSymptoms of endometriosis include debilitating pelvic pain, men-\nstrual disorders, dysmenorrhea, and dyspareunia, all of which nega-\ntively affect quality of life [2]. It has been estimated that endometrio-\nsis affects approximately 10% of premenopausal women, and 30% \nto 50% of women with endometriosis experience infertility [3].\nThe diagnosis of endometriosis is often delayed because it relies \non laparoscopy [4]. Current treatment options are limited to invasive \nsurgery or medications that suppress estrogen production. These \napproaches are primarily directed toward symptom relief rather than \ndefinitive treatment and may have unfavorable effects on fertility [5].\nLactoferrin is a glycoprotein with multiple biological activities, in-\ncluding anti-inflammatory, antioxidant, antimicrobial, and anticancer \neffects. It also has iron-binding and immunomodulatory properties \n[6,7]. Lactoferrin is present at high concentrations in colostrum and is \nalso found in exocrine secretions such as milk, saliva, tears, and gas-\n\ntrointestinal secretions. In addition, it is localized within the second-\nary granules of neutrophils [8,9].\nEndometriosis-associated subfertility is likely multifactorial and \nmay involve a disrupted pelvic microenvironment, inflammatory cy-\ntokines, altered redox status, and disturbed iron balance. These ab-\nnormalities may adversely affect fertilization and implantation and \nimpair oocyte and embryo quality [10,11]. Given its iron-binding ca-\npacity and potent anti-inflammatory and antioxidant properties, lac-\ntoferrin may represent a promising non-hormonal therapeutic can-\ndidate for improving fertility outcomes in endometriosis [12,13].\nNevertheless, evidence regarding the impact of lactoferrin on en-\ndometriosis-associated subfertility remains limited. Therefore, the \npresent study aimed to evaluate the potential ameliorative role of \nlactoferrin in experimentally induced endometriosis in rats by assess-\ning reproductive performance and related histopathological and en-\ndocrine alterations.\nMethods\n1. Animals\nForty mature female Sprague-Dawley rats (8 to 10 weeks old; 170 \nto 200 g) were purchased from the Helwan Breeding Farm of the \nEgyptian Organization for Vaccine and Biological Preparations. The \nanimals were housed in stainless steel cages in the animal house of \nthe Zoology Department, Faculty of Science, Mansoura University, \nunder standard conditions (22±2 °C; 12:12-hour light/dark cycle) \nwith free access to standard chow and water. Approval to conduct \nthe study was obtained from the Ethical Research Project Committee \nat Mansoura University (ACUC.SC.PhD.23.07.12).\n2. Experimental groups\nDaily vaginal smears were evaluated to determine the estrous cy-\ncle stage of each rat, and only rats in estrus were included in the \nstudy. Smears were assessed according to standard cytological crite-\nria, specifically the predominance of cornified epithelial cells. After 1 \nweek of acclimatization, rats were randomly divided into four groups, \neach comprising 10 animals: group I, Sham-operated rats; group II, \nSham rats treated with lactoferrin (Sham+Lacto); group III, endome-\ntriosis control rats (Endo); and group IV, endometriotic rats treated \nwith lactoferrin (Endo+Lacto). Randomization was performed using \nthe envelope method, and investigators performing outcome as-\nsessment were blinded to group allocation.\n3. Induction of endometriosis\nAccording to the modified method of Vernon and Wilson [14], rats \nin the Endo and Endo+Lacto groups that were in estrus were used to \nestablish the surgically induced endometriosis model. Thirty minutes \nafter confirmation of estrus, the rats were anesthetized by intraperi-\ntoneal injection of a mixture of xylazine hydrochloride (5 mg/kg; Xy-\nlajet 20 mg/mL; ADWIA) and ketamine hydrochloride (75 mg/kg; \nKetamax 50 mg/mL; Troikaa Pharmaceuticals Ltd.). The rats were \nplaced on a heating pad to maintain body temperature at 37 °C. The \nventral abdomen was aseptically prepared from the xiphoid carti-\nlage to the pubic region and both lateral abdominal sides. A 5-cm \nmidline incision was made in the ventral mesogastric region overly-\ning the uterine horns. The left uterine horn was ligated, and an ap-\nproximately 2-cm segment was resected. The resected tissue was \nopened longitudinally and immersed in sterile saline. It was then cut \ninto four 5-mm² pieces, which were sutured to the inner abdominal \nwall using a simple interrupted pattern with 4-0 polyglactin 910 su-\nture (Vicryl; Ethicon), with the endometrial surface facing the ab -\ndominal cavity. The abdominal layers were closed using a simple \ncontinuous pattern with 4-0 polyglactin 910 suture. The subcutane-\nous layer was closed with a subcutaneous suture pattern using 4-0 \npolyglactin 910 suture, and the skin was closed with a simple inter-\nrupted pattern using 2-0 silk suture. After surgery, rats were housed \nindividually and received intramuscular ampicillin (15 mg/200 g) \nonce daily for 3 consecutive postoperative days. Postoperative anal-\ngesia consisted of intramuscular meloxicam (4 mg/kg) administered \nfor 3 days.\n4. Confirmation of implants and treatment protocol\nFour weeks postoperatively, a second abdominal surgery was per-\nformed to confirm the development of endometriotic implants. Suc-\ncessful model establishment was confirmed by the presence of visi-\nble, vascularized endometriotic implants at the second laparotomy. \nFive days after the second surgery, rats in the Endo+Lacto group re-\nceived oral lactoferrin at a dose of 300 mg/kg/day for 30 consecutive \ndays [15]. Lactoferrin was administered as a commercially available \noral preparation (Pravotin Lactoferrin-100 sachets; HYGINT). The con-\ntents of each sachet were dissolved in distilled water to prepare a 75 \nmg/mL solution, such that 1 mL contained 75 mg of lactoferrin, and \nthe solution was administered by oral gavage.\nThe lactoferrin dose (300 mg/kg/day) was selected on the basis of \nprevious experimental studies indicating its safety and efficacy after \noral administration in rats, together with preliminary pilot observa-\ntions [16,17]. A 30-day treatment period was selected to encompass \nmultiple estrous cycles and thereby allow adequate assessment of \nhormonal changes, reproductive tissue remodeling, and pregnancy \noutcomes in rats [18].\nThe Sham-operated and Sham+Lacto groups underwent the \nsame anesthesia, abdominal incision, and suturing procedures, using \nthe same suture patterns and materials described above, but with-\nout manipulation of the uterus. Five days after surgery, the Sham+-\nhttps://doi.org/10.5653/cerm.2026.091662\nClin Exp Reprod Med [Epub ahead of print]\n\nLacto group received oral lactoferrin at 300 mg/kg/day for 30 days.\n5. Sample collection\nFive rats from each group were euthanized after the treatment pe-\nriod by intraperitoneal injection of thiopental sodium (50 mg/kg; 0.5 \ng thiopental, EIPCO). Immediately before euthanasia, vaginal smears \nwere obtained, and all animals were confirmed to be in estrus to \nminimize cycle-related variability in gonadotropin levels. Blood sam-\nples were collected by cardiac puncture, centrifuged at 3,000 rpm for \n10 minutes to separate serum, and stored at −20 °C for subsequent \nanalysis. Eutopic uterine tissues and ovaries were excised and fixed in \n10% neutral formalin for histopathological evaluation.\n1) Body weight\nBody weights (g) of all rats were recorded at the beginning and \nend of the experimental period.\n2) Determination of hormone levels\nSerum follicle-stimulating hormone (FSH) and luteinizing hor -\nmone (LH) levels were measured using rat enzyme-linked immuno-\nsorbent assay kits supplied by CUSABIO (catalog numbers CSB-\nE06869r and CSB-E12654r, respectively), according to the manufac-\nturer’s instructions.\n3) Histological examinations\nEutopic uterine and ovarian tissues were fixed in 10% neutral for-\nmalin and then processed through dehydration, clearing, paraffin \nembedding, sectioning, and hematoxylin and eosin (H&E) staining. \nSections (5 µm) were examined under a bright-field Olympus light \nmicroscope equipped with an AmScope MU1000 camera at 100× \nand 400× magnification [19].\n6. Fertility index determination\nFollowing the treatment period, five females from each group \nwere housed with mature males at a ratio of one male to two fe -\nmales. The males were proven fertile (8 to 10 weeks old; 200 to 230 \ng), and cohabitation continued for 14 days. Males were randomly as-\nsigned across groups to minimize the influence of male fertility on \nreproductive outcomes. Mating was confirmed by the presence of \nsperm in vaginal smears. Pregnant females were then housed indi-\nvidually until delivery.\nThree months after the initial endometriosis induction surgery, \nand after delivery, the dams underwent terminal dissection, and the \nmaternal ovaries and uteri were excised. Corpora lutea, identified as \nraised opaque structures on the ovarian surface, were counted using \na magnifying lens. In addition, implantation sites, recognized as pla-\ncental scars in the uterus, were counted. Fertility potential (implanta-\ntion index) was calculated as (number of implantation sites/number \nof corpora lutea)×100 [20,21]. Maternal ovarian and uterine weights \nwere recorded. Litter number and pup size, length, and weight were \nalso recorded. Selection of the five females per group for fertility test-\ning was random, and outcome assessment was performed with in-\nvestigators blinded to group allocation.\n7. Reproductive performance indices\nReproductive performance indices were calculated as follows: \nmating index (%)=(number of females mated/number of females \nplaced with males)×100; pregnancy index (%)=(number of females \npregnant/number of females mated)×100; preimplantation loss \n(%)=[(number of corpora lutea−number of implantation sites)/num-\nber of corpora lutea]×100; and postimplantation loss (%)=[(number \nof implantation sites−number of neonates)/number of implantation \nsites]×100.\n8. Statistical analysis\nGraphPad Prism ver. 8 (GraphPad Software Inc.) was used for data \nanalysis. Normality was assessed using the Shapiro-Wilk test. Results \nare presented as mean±standard error (SE). For neonatal outcomes, \nthe litter was considered the experimental unit. Individual pup mea-\nsurements, including pup weight, length, and size, were averaged \nwithin each litter, and each litter/dam contributed one data point to \nthe analysis. Litter size, defined as the number of pups per litter, was \nrecorded once for each dam and was also analyzed using the litter as \nthe experimental unit. Group comparisons were performed using \none-way analysis of variance followed by the Tukey post hoc test. A \np-value <0.05 was considered statistically significant.\nResults\n1. Body-weight change\nFigure 1 shows no significant differences in baseline or final body \nweight among the groups. Lactoferrin administration to Sham rats \ndid not significantly affect body-weight change compared with \nSham controls. In contrast, the Endo group showed a significantly \nsmaller body-weight change than the Sham group (p≤0.01). Lacto-\nferrin treatment did not significantly affect body-weight change in \nendometriotic rats compared with the Endo group.\n2. Follicle-stimulating hormone\nAs shown in Figure 2, lactoferrin administration to Sham rats did \nnot significantly alter serum FSH levels compared with Sham con-\ntrols. The Endo group exhibited a significant elevation in serum FSH \nlevels compared with the Sham group (p≤0.001). Lactoferrin treat-\nment significantly reduced serum FSH levels in endometriotic rats \nwww.eCERM.org 3\nBaky MMA et al. Lactoferrin and endometriosis experimental study\n\nFigure 2. Follicle-stimulating hormone (FSH) levels in the different \ngroups. Data are presented as mean±standard error of the mean \n(n=5). \na)\np≤0.01; \nb)\np≤0.001; \nc)\nSignificant compared with the Sham \ngroup; \nd)\nSignificant compared with the Endo group.\nFigure 3. Luteinizing hormone (LH) levels in the different groups. \nData are presented as mean±standard error of the mean (n=5).  \na)\np≤0.001; \nb)\nSignificant compared with the Sham group; \nc)\nSignificant \ncompared with the Endo group.\nFigure 1. Initial body weight (BW), final BW, and BW change in the \ndifferent groups. Data are presented as mean±standard error of the \nmean (n=10 rats/group). \na)\np≤0.01 compared with the Sham group.\na**\n0\n50\n100\n150\n200\n250\nSham Sham-Lacto Endo Endo+Lacto\nBody Weight (g)\nAnimal groups\nInitial BW Final BW BW change\na)\nSham \nSham-Lacto\nEndo\nEndo+Lacto\n0\n5\n10\n15\n20\n25\nAnimal groups\nLH (ng / mL)\nb***\na***\na***b***\na),b)\na),c)\na),b),c)\nBody weight (g)\nFSH (ng/mL)\nLH (ng/mL)\nSham+Lacto\ncompared with the Endo group (p≤0.001).\n3. Luteinizing hormone\nAs shown in Figure 3, lactoferrin administration to Sham rats did \nnot significantly alter serum LH levels compared with Sham controls. \nThe Endo group showed a significant decrease in serum LH levels \ncompared with the Sham group ( p≤0.001). Lactoferrin treatment \nsignificantly increased serum LH levels in endometriotic rats com-\npared with the Endo group (p≤0.001).\n4. Histopathological findings\nAs shown in Figure 4, H&E-stained uterine sections from the Sham \ngroup demonstrated normal eutopic uterine architecture, including \na simple columnar epithelium lining the uterine lumen and a cellular \nstroma containing tubular glands lined by regular columnar epitheli-\nal cells. The Sham+Lacto group displayed uterine histology compa-\nrable to that of the Sham group. In contrast, the Endo group showed \nhistopathological alterations, including fewer endometrial glands \nwithin the stroma and epithelial degeneration with cytoplasmic vac-\nuolation. Lactoferrin treatment improved uterine histology in endo-\nmetriotic rats compared with the Endo group.\nAs shown in Figure 5, ovarian sections from the Sham group \ndemonstrated normal ovarian architecture, including normal stromal \ncells, follicles at various stages of maturation, and corpora lutea. The \nSham+Lacto group showed ovarian histology comparable to that of \nthe Sham group. In contrast, the Endo group exhibited histopatho-\nlogical changes, including reduced numbers of ovarian follicles \nacross developmental stages and vacuolated stromal cells. Lactofer-\nrin treatment ameliorated these ovarian histopathological alterations \ncompared with the Endo group.\n5. Fertility index findings\n1) Maternal organ weights\nAs shown in Figure 6, lactoferrin administration to Sham rats did \nnot significantly affect maternal uterine or ovarian weights com -\npared with Sham controls. Compared with the Sham group, the \nEndo group exhibited significantly lower maternal uterine and ovari-\nan weights (p≤0.05). Lactoferrin treatment produced a nonsignifi-\ncant increase in maternal uterine and ovarian weights compared \nwith the Endo group.\n2) Fertility potential\nAs shown in Figure 7, lactoferrin administration to Sham rats did \nnot significantly affect fertility potential compared with Sham con-\nsham \nSham-Lacto\nEndo\nEndo+Lacto\n0\n5\n10\n15\n20\nAnimal groups\nFSH (ng / mL)\na***\na**b***\nb***\nc),d)\nb),d)\na),b),c),d)\nSham+Lacto Sham+Lacto\nhttps://doi.org/10.5653/cerm.2026.091664\nClin Exp Reprod Med [Epub ahead of print]\n\nFigure 4. (A) Eutopic endometrial tissue in the Sham group showed normal histological architecture, with a regular simple columnar \nepithelium (E) lining the lumen (L) and a highly cellular stroma (S) containing tubular endometrial glands (G). (B) Eutopic endometrial tissue \nin the Sham+Lacto group showed a normal structure, with the uterine L lined by regular endometrial E and the endometrial S containing \ntubular endometrial G. (C) Eutopic endometrial tissue in the Endo group showed damaged endometrial E lining the uterine L, along with S \ncontaining fewer endometrial G. (D) Eutopic endometrial tissue in the Endo+Lacto group showed improved uterine histology compared with \nthe Endo group, with endometrial E lining the L and S containing well-differentiated endometrial G (hematoxylin and eosin, ×100 and ×400).\nFigure 4\ntrols. The Endo group showed no significant reduction in fertility po-\ntential compared with the Sham group (p≥0.05). Lactoferrin treat-\nment produced a trend toward increased fertility potential compared \nwith the Endo group; however, this change did not reach statistical \nsignificance.\n3) Neonatal outcomes\nAs shown in Figure 8, lactoferrin administration to Sham rats did \nnot significantly affect neonatal weight, length, size, or pup number \ncompared with Sham controls. Compared with the Sham group, the \nEndo group showed no significant differences in these neonatal out-\ncomes (p≥0.05). Compared with the Endo group, lactoferrin treat-\nment significantly increased neonatal weight and size in endometri-\notic rats (p≤0.01), whereas increases in neonatal length and pup \nnumber were not statistically significant. These data are presented in \nTable 1.\nDiscussion\nEndometriosis is a chronic, inflammatory, hormone-dependent \nwww.eCERM.org 5\nBaky MMA et al. Lactoferrin and endometriosis experimental study\nAA\nBB\nCC\nDD\n\nFigure 5\nFigure 5. (A) Ovarian tissue in the Sham group showed normal histological architecture, including primary follicles (PF), secondary follicles (SF), \nGraafian follicles (GF), well-developed corpora lutea (CL), and blood vessels (BV). (B) Ovarian tissue in the Sham+Lacto group showed normal \nstructure, with several ovarian follicles, including PF , SF , GF , and CL. (C) Ovarian tissue in the Endo group showed only PF and CL, along with \nsome vacuolated stromal cells (arrowheads). (D) Ovarian tissue in the Endo+Lacto group showed improved ovarian histology compared with \nthe Endo group, with several ovarian follicles, including PF , SF , and CL (hematoxylin and eosin, ×100 and ×400).\nTable 1. Reproductive performance indices in the studied groups\nNeonatal outcome Sham group Sham+Lacto group Endo group Endo+Lacto group\nNo. of females placed with males 5 5 5 5\nNo. of females mated 5 5 5 5\nNo. of females pregnant 5 5 5 5\nMating index (%) 100 100 100 100\nPregnancy index (%) 100 100 100 100\nPreimplantation loss (%) 15.63 ± 15.63 9.375 ± 3.375 41.68 ± 9.765 6.25 ± 6.25\nPostimplantation loss (%) 0 0 0 0\nValues are presented as mean±standard error. Data were analyzed using one-way analysis of variance followed by Tukey’s post hoc test. No statistically signif-\nicant differences were detected among the groups.\nhttps://doi.org/10.5653/cerm.2026.091666\nClin Exp Reprod Med [Epub ahead of print]\nAA\nBB\nCC\nDD\n\nFigure 6. Maternal (A) ovarian weight and (B) uterine weight in the different groups. Data are presented as mean±standard error of the mean \n(n=5 dams/group). \na)\np≤0.05 compared with the Sham group.\nFigure 7.  Fertility potential in the different groups. Data are \npresented as mean±standard error of the mean (n=5 dams/group). \nNo statistically significant differences were detected among the \ngroups.\na)\na)\nSham\nSham-Lacto\nEndo\nEndo+Lacto\n0\n50\n100\n150\nAnimal groups\nFertility potential (%)\nOvary weight (g)\nOvary weight (g)\ncondition that negatively affects fertility [22]. Lactoferrin is a multi-\nfunctional glycoprotein with anti-inflammatory, antioxidant, and im-\nmunomodulatory properties [23]. Accordingly, the present study \nevaluated the potential ameliorative effect of lactoferrin on endome-\ntriosis-associated subfertility in an experimentally induced rat model \nof endometriosis.\nIn the present study, serum FSH levels were significantly elevated, \nwhereas LH levels were significantly decreased, in endometriosis rats \ncompared with Sham rats. These hormonal disturbances are consis-\ntent with previous reports suggesting that disruption of the hypo-\nthalamic-pituitary-ovarian axis may result from the inflammation \nand hormonal imbalance associated with endometriosis, leading to \novulatory and endometrial abnormalities [10,24].\nThe histopathological findings of the present study support these \nhormonal changes. Ovarian tissue from the Endo group showed de-\ngenerative alterations in the ovarian cortex, together with reduced \nnumbers of developing follicles, compared with the Sham group. \nThese findings are consistent with previous reports indicating that \nperitoneal oxidative stress and inflammation may disrupt folliculo-\ngenesis and ovulation, thereby reducing ovarian reserve [25,26]. Fur-\nthermore, the impaired oocyte quality associated with endometrio-\nsis may reduce embryo quality and implantation potential [27].\nIn addition, the present study demonstrated histopathological al-\nterations in the eutopic endometrium of Endo rats that may adverse-\nly affect implantation. These findings are consistent with previous re-\nports indicating that the hormonal imbalance, altered redox status, \nand inflammatory mediators associated with endometriosis may in-\nduce structural and functional changes in the eutopic endometrium, \nthereby impairing implantation or fetal growth [27,28]. Moreover, \nprogesterone resistance in endometriosis has been associated with \nluteal phase defects that may negatively affect endometrial receptiv-\nity [29].\nThe present results showed that maternal reproductive organ \nweights were significantly reduced in Endo rats compared with \nSham rats, whereas fertility-related parameters showed only a non-\nsignificant downward trend. Although implantation efficiency did \nnot differ significantly among groups, the improved neonatal out-\ncomes observed in the Endo+Lacto group suggest that lactoferrin \nmay improve the endometrial and fetal developmental environment \n[30,31].\nThese findings are consistent with reports indicating that perito-\nneal inflammation, redox imbalance, altered hormonal secretion, im-\nmune dysfunction, and pelvic anatomical changes associated with \nendometriosis may adversely affect ovulation, oocyte transport, \nsperm function, embryo development, and implantation potential \n[10,11,32]. Notably, lactoferrin treatment ameliorated gonadotropin \nimbalance, alleviated ovarian and eutopic endometrial histopatho-\nwww.eCERM.org 7\nBaky MMA et al. Lactoferrin and endometriosis experimental study\nAA BB\n\nFigure 8. Neonatal outcomes: (A) mean pup weight per litter, (B) mean pup length per litter, (C) mean pup size per litter, and (D) number of \npups per litter in the different groups. Individual pup measurements were averaged within each litter, and each litter was considered one \nexperimental unit. Data are presented as mean±standard error of the mean (n=5 L/group; 1 L per dam). \na)\np≤0.01 compared with the Endo \ngroup.\na)\na)\nNeonates weight (g)Neonates size (mm\n2\n)\nNeonates lenght (cm)Neonates number\nlogical alterations, and improved several fertility-related parameters \nin Endo rats. These ameliorative effects of lactoferrin may be attribut-\nable, at least in part, to its anti-inflammatory, antioxidant, and immu-\nnomodulatory properties, consistent with previous reports [33-35].\nAlthough the present study did not directly assess inflammatory \ncytokines, oxidative stress biomarkers, or immune cell infiltration, the \nobserved improvement in gonadotropin balance and reproductive \ntissue histology indirectly supports the proposed anti-inflammatory \nand antioxidant effects of lactoferrin, consistent with previous litera-\nture [6,36].\nLactoferrin is a glycoprotein with minimal reported toxicity [37]. It \nhas also been evaluated under the U.S. Food and Drug Administra-\ntion Generally Recognized as Safe notification program for use as a \nfood ingredient [ 38]. In addition, it may represent a potential \nnon-hormonal therapeutic candidate for endometriosis compared \nwith currently used medical therapies, such as combined oral contra-\nceptives, progestins, and gonadotropin-releasing hormone agonists/\nantagonists, which rely primarily on hormonal suppression. Al -\nthough such therapies may provide symptom relief, they are associ-\nated with hypoestrogenic adverse effects and are generally unsuit-\nable for women attempting conception [11,39].\nMoreover, given lactoferrin’s favorable safety profile in clinical and \nnutritional research, dose extrapolation based on body surface area \nsuggests that the selected dose falls within a biologically relevant \nand safe range for human oral supplementation [13,40].\nThe present study has several limitations. First, the sample size \nused for fertility evaluation was relatively small, and direct evaluation \nof ovarian reserve markers, such as anti-Müllerian hormone (AMH) \nlevels and follicle count, as well as inflammatory and oxidative stress \nbiomarkers, was not performed. Second, histological evaluation was \nmainly qualitative, and quantitative follicle counts and endometrial \nmorphometry were not included. Therefore, although the present \nresults support improvements in selected reproductive and neonatal \noutcomes, direct evaluation of AMH and follicle count is required be-\nfore definitive conclusions can be drawn regarding changes in ovari-\nan reserve. To better elucidate the mechanistic pathways underlying \nlactoferrin-mediated amelioration, future studies with larger cohorts \nand direct assessment of inflammatory cytokines, oxidative stress \nhttps://doi.org/10.5653/cerm.2026.091668\nClin Exp Reprod Med [Epub ahead of print]\nAA\nCC\nBB\nDD\n\nmarkers, and immune cell infiltration in ectopic and eutopic tissues \nare warranted.\nIn conclusion, although lactoferrin did not significantly restore im-\nplantation efficiency, it improved postimplantation pregnancy-relat-\ned outcomes, as reflected by increased neonatal weight and size. \nOverall, the present findings suggest that lactoferrin mitigates sever-\nal endocrine and histopathological alterations associated with endo-\nmetriosis in a rat model, including improved gonadotropin balance \nand amelioration of ovarian and eutopic endometrial histology. \nThese findings support lactoferrin as a potential non-hormonal ther-\napeutic candidate for improving selected pregnancy-related out-\ncomes under endometriotic conditions, possibly through anti-in-\nflammatory and antioxidant actions. However, further experimental \nand clinical studies incorporating comprehensive fertility assess -\nment, ovarian reserve markers (e.g., AMH and quantitative follicle \ncounts), and direct measurement of inflammatory and oxidative \nstress biomarkers are required to confirm these findings, clarify the \nunderlying mechanisms, and strengthen their translational rele -\nvance.\nConflict of interest\nNo potential conflict of interest relevant to this article was report-\ned.\nORCID\nMona M. Abdel Baky https://orcid.org/0000-0001-6484-784X\nMarwa Abass https://orcid.org/0000-0003-2749-2693\nAuthor contributions\nConceptualization: MMAB, MA, HAEG. Methodology: MMAB, MA, \nHAEG. Formal analysis: MMAB, MA, HAEG. Data curation: MMAB, \nMEA, MA, MAE. Visualization: MMAB, MEA, MA, MAE. Software: \nMMAB, MEA, MA. Validation: MMAB, MEA, MA. Investigation: MMAB, \nMEA, MA. Supervision: MMAB, MA, HAEG. Writing-original draft: \nMMAB, MA. Writing-review & editing: MMAB, MA. Approval of final \nmanuscript: MMAB, MEA, MA, MAE, HAEG.\nReferences\n1. Park W, Lim W, Kim M, Jang H, Park SJ, Song G, et al. Female repro-\nductive disease, endometriosis: from inflammation to infertility. \nMol Cells 2025;48:100164.\n2. Boucher A, Brichant G, Gridelet V, Nisolle M, Ravet S, Timmermans \nM, et al. Implantation failure in endometriosis patients: etiopatho-\ngenesis. J Clin Med 2022;11:5366.\n3. Colombo GE, Salmeri N, Leonardi M. Integrating IVF and surgical \nmanagement in endometriosis-associated infertility: a review. \nAdv Ther 2026;43:1004-15.\n4. Simancas-Racines D, Jimenez-Flores E, Montalvan M, Horowitz R, \nAraujo V, Reytor-Gonzalez C. Endometriosis as a systemic and \ncomplex disease: toward phenotype-based classification and \npersonalized therapy. Int J Mol Sci 2026;27:908.\n5. Saunders PT, Horne AW. Endometriosis: etiology, pathobiology, \nand therapeutic prospects. Cell 2021;184:2807-24.\n6. Demir R, Saritas S, Bechelany M, Karav S. Lactoferrin: properties \nand potential uses in the food industry. Int J Mol Sci \n2025;26:1404.\n7. Du Y, Li D, Chen J, Li YH, Zhang Z, Hidayat K, et al. Lactoferrin im-\nproves hepatic insulin resistance and pancreatic dysfunction in \nhigh-fat diet and streptozotocin-induced diabetic mice. Nutr Res \n2022;103:47-58.\n8. Belles A, Aguirre-Ramirez D, Abad I, Parras-Molto M, Sanchez L, \nGrasa L. Lactoferrin modulates gut microbiota and Toll-like recep-\ntors (TLRs) in mice with dysbiosis induced by antibiotics. Food \nFunct 2022;13:5854-69.\n9. Rizzi M, Manzoni P , Germano C, Quevedo MF , Sainaghi PP . Lacto-\nferrin, a natural protein with multiple functions in health and dis-\nease. Nutrients 2025;17:3403.\n10. Qi Q, Li Y, Chen Z, Luo Z, Zhou T, Zhou J, et al. Update on the patho-\ngenesis of endometriosis-related infertility based on contempo-\nrary evidence. Front Endocrinol (Lausanne) 2025;16:1558271.\n11. Elizur SE, Mostafa J, Berkowitz E, Orvieto R. Endometriosis and in-\nfertility: pathophysiology, treatment strategies, and reproductive \noutcomes. Arch Gynecol Obstet 2025;312:1037-48.\n12. Nakamura A, Tanaka Y, Tsuji S, Amano T, Takebayashi A, Takahashi \nA, et al. Bovine lactoferrin suppresses the proliferation of endo-\nmetriotic stromal cells via the PI3K/Akt/mTOR pathway. Biochem \nCell Biol 2025;103:1-8.\n13. Kruzel ML, Zimecki M, Actor JK. Lactoferrin in a context of inflam-\nmation-induced pathology. Front Immunol 2017;8:1438.\n14. Vernon MW, Wilson EA. Studies on the surgical induction of endo-\nmetriosis in the rat. Fertil Steril 1985;44:684-94.\n15. Elazab MF , Elbaiomy AE, Ahmed MS, Alsharif KF , Dahran N, Elma-\nhallawy EK, et al. Ameliorative effects of bovine lactoferrin on \nbenzene-induced hematotoxicity in albino rats. Front Vet Sci \n2022;9:907580.\n16. Abd El-Rahman SS, Ashwish NM, Ali ME. Appraisal of the \npre-emptive effect of lactoferrin against chromium-induced tes-\nticular toxicity in male rats. Biol Trace Elem Res 2023;201:5321-34.\n17. Asaad GF , Mostafa RE. Lactoferrin mitigates ethanol-induced gas-\ntric ulcer via modulation of ROS/ICAM-1/Nrf2 signaling pathway \nwww.eCERM.org 9\nBaky MMA et al. Lactoferrin and endometriosis experimental study\n\nin Wistar rats. Iran J Basic Med Sci 2022;25:1522-7.\n18. Goldman JM, Murr AS, Cooper RL. The rodent estrous cycle: char-\nacterization of vaginal cytology and its utility in toxicological \nstudies. Birth Defects Res B Dev Reprod Toxicol 2007;80:84-97.\n19. Anggorowati N, Ratna Kurniasari Ch, Damayanti K, Cahyanti T, \nWidodo I, Ghozali A, et al. Histochemical and immunohistochem-\nical study of α-SMA, collagen, and PCNA in epithelial ovarian neo-\nplasm. Asian Pac J Cancer Prev 2017;18:667-71.\n20. Haeri S, Minaie B, Amin G, Nikfar S, Khorasani R, Esmaily H, et al. \nEffect of Satureja khuzestanica essential oil on male rat fertility. Fi-\ntoterapia 2006;77:495-9.\n21. Parandin R, Yousofvand N, Ghorbani R. The enhancing effects of \nalcoholic extract of Nigella sativa seed on fertility potential, plas-\nma gonadotropins and testosterone in male rats. Iran J Reprod \nMed 2012;10:355-62.\n22. Cheng J, Li C, Ying Y, Lv J, Qu X, McGowan E, et al. Metformin alle-\nviates endometriosis and potentiates endometrial receptivity via \ndecreasing VEGF and MMP9 and increasing leukemia inhibitor \nfactor and HOXA10. Front Pharmacol 2022;13:750208.\n23. Cui S, Lv X, Sun G, Wu W, Xu H, Li Y , et al. Recent advances and \nprospects in purification and heterologous expression of lactofer-\nrin. Food Bioeng 2022;1:58-67.\n24. de Carvalho BR, Rosa-e-Silva AC, Rosa-e-Silva JC, dos Reis RM, \nFerriani RA, Silva-de-Sa MF . Increased basal FSH levels as predic-\ntors of low-quality follicles in infertile women with endometriosis. \nInt J Gynaecol Obstet 2010;110:208-12.\n25. Lee D, Kim SK, Lee JR, Jee BC. Management of endometriosis-re-\nlated infertility: considerations and treatment options. Clin Exp \nReprod Med 2020;47:1-11.\n26. Mansour G, Sharma RK, Agarwal A, Falcone T. Endometriosis-in-\nduced alterations in mouse metaphase II oocyte microtubules \nand chromosomal alignment: a possible cause of infertility. Fertil \nSteril 2010;94:1894-9.\n27. Kanellopoulos D, Karagianni D, Pergialiotis V, Nikiteas N, Lazaris \nAC, Iliopoulos D. Endometriosis and subfertility: a literature re-\nview. Maedica (Bucur) 2022;17:458-63.\n28. Leone Roberti Maggiore U, Chiappa V, Ceccaroni M, Roviglione G, \nSavelli L, Ferrero S, et al. Epidemiology of infertility in women \nwith endometriosis. Best Pract Res Clin Obstet Gynaecol 2024; \n92:102454.\n29. Atsama PM, Djiogue S, Awounfack CF , Njamen D. Graptophyllum \npictum (Acanthaceae) relieves some hallmarks of endometriosis in \nan experimental model in Wistar rats. Reprod Fertil 2025;6:e240084.\n30. Hashem NA, Wehaish FE, Elseady YY . Immunomodulatory effect \nof lactoferrin on mucosal immunity of uterus in pregnant rat. \nBiomed J Sci Tech Res 2021;37:29787-99.\n31. Messina A, El Motarajji S, Masturzo B, Manzoni P . The role of lacto-\nferrin in modulating inflammation and preventing preterm birth: \na narrative review. Nutrients 2025;17:3164.\n32. Moise A, Dzeitova M, de Landsheere L, Nisolle M, Brichant G. En-\ndometriosis and infertility: gynecological examination practical \nguide. J Clin Med 2025;14:1904.\n33. Li S, Liu M, Ma H, Jin Q, Ma Y, Wang C, et al. Ameliorative effect of re-\ncombinant human lactoferrin on the premature ovarian failure in \nrats after cyclophosphamide treatments. J Ovarian Res 2021;14:17.\n34. Sato B, Kanai S, Sakaguchi D, Yajima K, Matsumoto Y , Morohoshi K, \net al. Suppressive role of lactoferrin in overweight-related female \nfertility problems. Nutrients 2022;14:938.\n35. Horiuchi Y, Higuchi T, Tatsumi K, Takakura K, Fujii S, Konishi I. Lac-\ntoferrin is associated with a decrease in oocyte depletion in mice \nreceiving cyclophosphamide. Fertil Steril 2009;91:2069-78.\n36. Kowalczyk P , Kaczynska K, Kleczkowska P , Bukowska-Osko I, \nKramkowski K, Sulejczak D. The lactoferrin phenomenon: a mira-\ncle molecule. Molecules 2022;27:2941.\n37. Yamauchi K, Toida T, Nishimura S, Nagano E, Kusuoka O, Teraguchi \nS, et al. 13-Week oral repeated administration toxicity study of \nbovine lactoferrin in rats. Food Chem Toxicol 2000;38:503-12.\n38. Yang S, Huang HC, Hsu YF , Chuang CY. Inactivation of bacterial \nand viral bioaerosols by lactoferricin B-coated filters under vari-\nous environmental conditions. Hygiene 2026;6:18.\n39. Becker CM, Bokor A, Heikinheimo O, Horne A, Jansen F , Kiesel L, et \nal. ESHRE guideline: endometriosis. Hum Reprod Open 2022;  \n2022:hoac009.\n40. Actor JK, Hwang SA, Kruzel ML. Lactoferrin as a natural immune \nmodulator. Curr Pharm Des 2009;15:1956-73.\nhttps://doi.org/10.5653/cerm.2026.0916610\nClin Exp Reprod Med [Epub ahead of print]","source_license":"public-domain-us","license_restricted":false}