Table 1.

Clinical studies with LF in the prophylaxis and therapy of women suffering from genital tract infection and inflammation.

Type of Study, Number of Participants, Country	Type of LF, Dose, Mode and Time of LF Application	Clinical/Laboratory Effects	References
Randomized, double-blind, placebo-controlled clinical study; n = 40 (women, healthy volunteers, of fertile age, 18–50 years); Italy	BLF RCXTM (50 mg) plus probiotics (5 × 109 CFU): L. acidophilus La-14 (ATCC SD5212) plus L. rhamnosus HN001 (AGAL NM07/09514) in Respecta® complex (Giellepi S.p.A. Health Science, Seregno, Italy) in p.o. capsule Placebo: identical capsule containing 100 mg maltodextrin; 2 capsules/day, after breakfast, for 14 consecutive days	Vaginal swabs, collected at weeks 0, 1, 2 and 3 and analyzed for the consumed microorganisms by qPCR; vaginal pH determined Vaginal L. acidophilus and L. rhamnosus levels on days 14 and 21 ↑ Transient vaginal colonization (at least double levels from baseline) L. acidophilus on days 14 and 21, in 12 and 16, respectively, out of 20 women Transient colonization of L. rhamnosus on days 7 and 21, in 16 and 17, respectively, out of 20 women Vaginal pH (4.06–4.24) → No significant change in placebo group No AEs neither correlated nor unrelated	[113]
Randomized, double-blind, placebo-controlled clinical study; n = 40 (women of fertile age, 18–50 years, with symptomatic vaginal dysbiosis, signs or symptoms of vaginitis/vaginosis); Italy	BLF RCXTM (50 mg) plus probiotics (5 × 109 CFU): L. acidophilus GLA-14 (LMG S-29159) plus L. rhamnosus HN001 (AGAL NM07/09514) in Respecta® complex (Giellepi S.p.A. Health Science, Lissone, Italy) in p.o. capsule Placebo: identical capsule containing 100 mg maltodextrin; 1 capsule/day, for 15 consecutive days	Vaginal swabs, collected at days 0 and 15 and analyzed for the consumed microorganisms by qPCR; symptoms, pH of vaginal secretions and Nugent score assessed through the study Symptoms (in particular, itching, vaginal discharge and fishy odor) ↓ Nugent score ↓ (from intermediate 5.0 to normal 2.9) Vaginal pH ↓ (from 4.42 to 4.09) Vaginal colonization of both probiotic strains at the end of the study ↑ No significant change in placebo group No AEs, neither correlated nor unrelated	[114]
Randomized, double-blind, placebo-controlled clinical study; n = 48 (adult women with recurrent BV, treated with metronidazole 500 mg twice daily for 7 days); Italy	BLF RCXTM (50 mg) plus probiotics (5 × 109 CFU): L. acidophilus GLA-14 (LMG S-29159) plus L. rhamnosus HN001 (ATCC SD5675) in Respecta® complex (Giellepi S.p.A. Health Science, Lissone, Italy) in p.o. capsule Placebo: identical capsule containing 100 mg maltodextrin; 2 capsules/day, for 5 consecutive days, followed by a further 10 consecutive days at a dosage of 1 capsule/day (induction phase) During the maintenance phase (6 months following the induction phase), 1 capsule/day, for 10 consecutive days per month, starting the first day of menstrual cycle (prophylactic treatment) since the menstrual blood increases the vaginal pH and contributes to increasing the risk of BV recurrences	Normalization of Nugent score, remission of symptoms, recurrences during a 6-month follow-up period were assessed Nugent score ↓ Recurrence rate ↓ Symptoms of BV (vaginal discharge and itching) ↓	[115]
n = 6 (5 pregnant women and 1 non-pregnant woman, with a history of multiple pregnancy losses or preterm delivery and refractory BV E. coli, Enterococcus, Gardnerella vaginalis, Staphylococcus); Japan	BLF (NRL Pharma, Kawasaki, Japan) in intravaginal suppositories, 150 mg/day and p.o. tablets, 700 mg/day, starting before pregnancy or from 11th–21st gestational week until delivery	Normalization of vaginal flora (appearance and gradual predominance of Lactobacillus) Patients achieved pregnancy and delivered at term No AEs in mothers and newborns	[116]
Open, prospective, randomized clinical study; n = 60 (sexually active women aged 18–45 years, with symptomatic acute BV); Italy	BLF (AG Pharma s.r.l. Rome, Italy), in vaginal tablets 100 mg or 200 mg, 1 tablet/day for 10 days	Outcomes were a clinical evaluation based on Amsel criteria and Nugent scores Vaginal pH and structure of the vaginal bacterial biota and its dynamics during the study, determined by culture-dependent and molecular-based techniques Vaginal pH ↓ (2 weeks after stopping treatment, 60% and 89% of patients had pH < 4.5 in 100 mg and 200 mg of BLF, respectively) Nugent score ↓ (2 weeks after stopping treatment, 43% and 75% of patients had Nugent score ≤ 3 in 100 mg and 200 mg of BLF, respectively) Occurrence of vaginal bacteria species associated with BV: Gardnerella, Prevotella and Lachnospira ↓ Occurrence of Streptococcus spp., Staphylococcus spp., Escherichia coli ↓ Occurrence of Candida spp. ↓ Occurrence of Lactobacillus species ↑ Bacterial biota balance was maintained up to 2 weeks after treatment, was stopped only in 200 mg BLF-treated patients	[117]
Single-center retrospective cohort clinical study; n = 125 (pregnant women in the first trimester with BV diagnosis, with a history of prior spontaneous preterm birth); Italy	LF * in vaginal tablets, 300 mg/day for 21 days	Incidence of preterm birth < 37 weeks of gestation was the primary outcome Rate of preterm birth ↓ Gestational age at delivery ↑ (37.7 weeks in LF group vs. 35.9 weeks in control group without LF) Rate of admission for threatened preterm labor ↓ (45% vs. 70.8%) No differences in other outcomes (chorioamnionitis, preterm PROM < 34 weeks and neonatal outcomes) No cases of late miscarriage were reported No AEs were reported	[118]
Case report; 38-year-old multiparous women with 3 preterm PROM, diagnosed as having refractory vaginitis (Streptococcus group B and Staphylococcus), not cured with estriol and antibiotics; Japan	BLF (NRL Pharma, Kawasaki, Japan) in intravaginal tablets, 150 mg/day, and p.o. tablets, 700 mg/day, for 41 weeks (13 weeks before pregnancy and 38 weeks after, until delivery)	Appearance of Lactobacillus in vaginal flora, the patient achieved pregnancy 3 months later and delivered a healthy infant After the delivery, LF application was discontinued and 1 and 3 months after, no Lactobacillus was detected in vaginal discharge cultures	[119]
Randomized, double-blind, placebo-controlled clinical study; n = 48 (women of fertile age, 18–50 years, with positive cultures for Candida spp., symptomatic acute episodes of VVC and with documented anamnestic history of recurrences confirmed by culture analysis, during the induction phase, being treated with standard antifungal therapy— clotrimazole 100 mg daily for 7 days); Italy	BLF RCXTM (50 mg) plus probiotics (5 × 109 CFU): L. acidophilus GLA-14 (LMG S-29159) plus L. rhamnosus HN001 (ATCC SD5675) in Respecta® complex (Giellepi S.p.A. Health Science, Lissone, Italy) in p.o. capsule Placebo: identical capsule containing 100 mg maltodextrin; 2 capsules/day, for 5 consecutive days, followed by a further 10 consecutive days at a dosage of 1 capsule/day (acute treatment) During the maintenance phase (6 months following the induction phase), 1 capsule/day for 10 consecutive days per month, in the premenstrual phase (prophylactic treatment) The timeline was related to the consideration that in the premenstrual (luteal) phase, the vagina becomes more vulnerable to the pathogens	Efficacy evaluation based on clinical overall cure rate: vaginal discharge or itching and negative cultures; recurrence rate during the 6-month follow-up period Itching ↓ (at 3 months, women without itching: 70.8% in the Respecta® group vs. 8.3% in the placebo group; at 6 months: 83.3% vs. 0%) Vaginal discharge ↓ (at 3 months women without discharge were 66.7% in Respecta® group vs. 8.3% in placebo group; at 6 months: 70.8% vs. 20.8%) Overall cure rate defined as the absence of any symptoms and yeasts indicated in culture from vaginal swabs ↓ (at 3 months: 66.7% vs. 8.3%; at 6 months: 70.8% vs. 0%) Recurrence rate was defined as the presence of any VVC symptoms and yeasts in culture from vaginal swabs ↓ (at 3 months: 33.3% vs. 91.7%; at 6 months: 29.2% vs. 100%)	[120]
n = 34 (women aged 25–45 years with signs and symptoms of acute VVC); Italy	BLF * 4% in vaginal cream, 5 g of cream in the vagina and 2 cm applied on the vulva twice a day for 7 days	Clinical and microscopic examination was performed Response to all the characteristic symptoms of VVC ↑ 27 patients completely recovered, 5 showed a good improvement, and 2 were still suffering from VVC at the end of treatment	[121]
Open-label cohort clinical study; n = 7 (pregnant women asymptomatically affected by Chlamydia trachomatis and showing a high concentration of IL-6 in cervical fluids); Italy	BLF, 20% iron-saturated (Morinaga Milk Ind., Tokyo, Japan), intravaginal, 100 mg every 8 h (daily doses 300 mg/person) for 30 days In vitro test on the anti-chlamydial effect of BLF: human epithelial HeLa-229 cell line was treated with 100 μg/mL BLF	In vivo test: C. trachomatis in cervicovaginal smears and IL-6 concentration in the cervical fluid were assessed 6 out of 7 cervical specimens were negative for C. trachomatis IL-6 in cervical fluids ↓ Patients achieved pregnancy and delivered at term No maternal and neonatal AEs In vitro test: inhibitory effect of BLF on C. trachomatis entry to cells, decrease in IL-6 and IL-8 levels induced by infection with C. trachomatis	[86]
One-center, placebo- controlled, cohort clinical study; n = 48 (pregnant women at risk of PROM); Egypt	rHLF * 100 mg, p.o. twice/day for 30 days before meals Placebo as control	Patient’s hospital stay → Duration of PROM to delivery → Mode of delivery (vaginal/cesarean section) → Risk of PROM ↓	[122]
Open-label, pilot clinical study; n = 21 (26–32 weeks pregnant women, suffering from IDA and abnormal vaginal flora, at risk of preterm delivery); Italy	BLF (Lattoferrina®, AG Pharma, Rome, Italy) p.o., 100 mg/day b.i.d. (daily doses 200 mg), before meals, every day for 1 month FeSO4 520 mg in p.o. tablets, as control	Outcomes were clinical characteristics and structure of the vaginal bacterial biota, determined by a culture-based method Normal vaginal microbiota ↑ (vaginal infection disappearance) Cervicovaginal IL-6 (vaginal inflammatory response) ↓ In both groups, cervical length and funneling (in the ultrasound data) did not change at follow-up after 10 and 30 days; pregnancy continued regularly, and all women had term delivery after 37 weeks	[123]
Randomized, open-label study; n = 60 (pregnant women undergoing genetic amniocentesis at the 16th gestational week, at risk of inflammation); Italy	BLF (Difesan®, Progine Farmaceutici, Firenze, Italy), 300 mg in vaginal tablet, once 4 h or 12 h prior amniocentesis	Amniotic IL-6 ↓	[124]
		Decreased levels of amniotic pro-inflammatory mediators: IL-9, IL-15, IFN-γ, IP-10, TNF-α, IL-1α, MCP-3, IL-2RA, IL-12p40, IFN-α2, IL-2, IL-4, eotaxin, PDGF-BB, RANTES, IL-18, MIF ↓ Increased levels of amniotic anti-inflammatory mediators: IL-17, FGF-b, G-CSF, GM-CSF, MCP-1, IL-3, SDF-1α ↑	[125]
	BLF (Difesan®, Progine Farmaceutici, Firenze, Italy), 300 mg in a vaginal tablet, once every 4 h or 12 h prior to amniocentesis In vitro test on the antioxidant effect of LTF: human monocytic U937 cell line was treated with 50 μg/mL LTF # for 4 h or 12 h	Decreased oxidative stress in vivo: Amniotic TBARS concentration ↓ Amniotic TAS ↑ Decreased oxidative stress in vitro: TBARS concentration ↓	[126]
Prospective, randomized study; n = 111 (pregnant women undergoing genetic amniocentesis at the 16–18th gestational week, at risk of inflammation); Italy	BLF (Difesan®, Progine Farmaceutici, Firenze, Italy), 300 mg in vaginal tablet, once every 4 h prior to amniocentesis	Regulation of the inflammatory markers in the amniotic fluid: PGE2, MMP-9 and TIMP-1 (inhibitor of MMP-1) ↓ MMP-2 ↑ TIMP-2 (inhibitor of MMP-2) →	[127]

AEs—adverse events; BLF—bovine lactoferrin; BV—bacterial vaginosis; CFU—colony forming units; FGFb—basic fibroblast growth factor; G-CSF—granulocyte colony-stimulating factor; GM-CSF—granulocyte-macrophage colony-stimulating factor; IFN—interferon; IL—interleukin; IP-10—interferon inducible protein; LF—lactoferrin; MCP-1—monocyte chemoattractant protein-1; MIF—macrophage migration inhibitory factor; MMP-2—matrix metalloproteinase-2; MMP-9—matrix metalloproteinase-9; OSI—oxidative stress index; PDGF—platelet-derived growth factor; PGE2—prostaglandin E2; PROM—premature rupture of membrane qPCR—quantitative polymerase chain reaction; RANTES—regulated on activation normal T cells expressed and secreted; rHLF—recombinant human LF; SDF-1α—stromal cell-derived factor 1α; TAS—total antioxidant status (expressed in Trolox equivalents or OSI); TBARS—thiobarbituric acid reactive substances (expressed as malondialdehyde—MDA equivalents); TIMP-1—tissue inhibitor of metalloproteinase-1; TIMP-2—tissue inhibitor of metalloproteinase; TNFα—tumor necrosis factor α; VVC—vulvovaginal candidiasis; * the authors did not identify what kind of LF preparation was used or only an abstract was available. ↑ increase, ↓decrease, → no change.