Dependency of mitochondrial quantity on blastocyst timeline obscures its actual effect to pregnancy outcomes.

Objectives: To explore the correlation between mitochondrial quantity and the blastocyst development timeline as well as their respective contributions to early pregnancy. Methods: A retrospective study was conducted using a dataset comprising 2,633 embryos that underwent preimplantation genetic testing for aneuploidy (PGT-A) between January 2016 and December 2023. The study was divided into three subsets to address distinct aspects: the representativeness of a single trophectoderm (TE) biopsy for mitochondrial quantity (n=43), the correlation between morphokinetic features and mitochondrial quantity (n=307), and the association analysis among mitochondrial quantity, blastocyst timeline factor, and reproductive outcomes (n=2,283). Distribution assessment of mitochondrial quantity across an individual blastocyst involved the identification within multiple biopsies and spent culture media. Timeline evaluation included correlating mitochondrial quantity with time-lapse datasets. Finally, multivariate logistic regression models, incorporating potential effectors alongside mitochondrial quantity, were employed to analyze their respective contributions to early pregnancy endpoints. Results: Of distribution assessment, mitochondrial quantity exhibited an even distribution across the entire trophectoderm (Spearman's ρ=0.82), while no detectable mtDNAs in the corresponding spent culture media. Then the timeline correlation study revealed significant association between mitochondrial quantity and blastocyst features of both the day of expanded blastocyst formation (95% Confidence intervals, CIs: 0.27~4.89, p=0.03) and the timing of expanded blastocyst formation (tEB) (95% CIs: -0.24~-0.01, p=0.04) in the regression model, indicating a strong dependency between mitochondrial quantity and the blastocyst development timeline. For the contribution to early pregnancy, multivariate logistic regression models showed that the day of expanded blastocyst formation contributed to four endpoints persistently: positive for HCG (odd ratio, OR: 0.71, p=0.006), gestational sac (OR: 0.78, p=0.04), fetal heartbeat (OR: 0.71, p=0.004), and progression to 14 weeks (OR: 0.69, p=0.002). Contrastingly, no notable correlation was observed between the mitochondrial quantity and these endpoints. Conclusions: Strong interaction was observed between mitochondrial quantity and the blastocyst timeline, particularly the timing of expanded blastocyst formation. It suggests that the primary determinant influencing pregnancy outcomes lies in the time-dependent parameter of blastocyst rather than in the specific mitochondrial quantity.

Structural and functional analyses of viral H2 protein of the vaccinia virus entry fusion complex.

IMPORTANCE: Vaccinia virus infection requires virus-cell membrane fusion to complete entry during endocytosis; however, it contains a large viral fusion protein complex of 11 viral proteins that share no structure or sequence homology to all the known viral fusion proteins, including type I, II, and III fusion proteins. It is thus very challenging to investigate how the vaccinia fusion complex works to trigger membrane fusion with host cells. In this study, we crystallized the ectodomain of vaccinia H2 protein, one component of the viral fusion complex. Furthermore, we performed a series of mutational, biochemical, and molecular analyses and identified two surface loops containing 170LGYSG174 and 125RRGTGDAW132 as the A28-binding region. We also showed that residues in the N-terminal helical region (amino acids 51-90) are also important for H2 function.

Characterization of the pH-dependent protein stability of 3α-hydroxysteroid dehydrogenase/carbonyl reductase by differential scanning fluorimetry.

The characterization of protein stability is essential for understanding the functions of proteins. Hydroxysteroid dehydrogenase is involved in the biosynthesis of steroid hormones and the detoxification of xenobiotic carbonyl compounds. However, the stability of hydroxysteroid dehydrogenases has not yet been characterized in detail. Here, we determined the changes in Gibbs free energy, enthalpy, entropy, and heat capacity of unfolding for 3α-hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) by varying the pH and urea concentration through differential scanning fluorimetry and presented pH-dependent protein stability as a function of temperature. 3α-HSD/CR shows the maximum stability of 30.79 kJ mol-1 at 26.4°C, pH 7.6 and decreases to 7.74 kJ mol-1 at 25.7°C, pH 4.5. The change of heat capacity of 30.25 ± 1.38 kJ mol-1 K-1 is obtained from the enthalpy of denaturation as a function of melting temperature at varied pH. Two proton uptakes are linked to protein unfolding from residues with differential pKa of 4.0 and 6.5 in the native and denatured states, respectively. The large positive heat capacity change indicated that hydrophobic interactions played an important role in the folding of 3α-HSD/CR. These studies reveal the mechanism of protein unfolding in HSD and provide a convenient method to extract thermodynamic parameters for characterizing protein stability using differential scanning fluorimetry.

Reduced mitochondrial DNA content correlate with poor clinical outcomes in cryotransfers with day 6 single euploid embryos.

Objective: To investigate whether the mitochondrial DNA (mtDNA) content of a single biopsy at trophoblast correlates with the developmental potential and reproductive outcomes of blastocyst. Methods: A retrospective analysis applied the dataset of 1,675 embryos with preimplantation genetic testing for aneuploidy (PGT-A) from 1,305 individuals, and 1,383 embryos involved cryotransfers of single euploid embryo between January 2015 and December 2019. The studied cohort was divided for algorithm establishment on the NGS platform (n=40), correlation of biological features (n=1,635), and correlation of reproductive outcomes (n=1,340). Of the algorithm derived from the NGS platform, the reliability and repeatability were validated via qPCR assay and inter-run controls, respectively. Of the correlation across biological features, stratification analyses were applied to evaluate the effect from a single contributor. Eventually, the correlation between the mtDNA ratios and reproductive outcomes was adjusted according to the significant effector(s). Results: The mtDNA ratios showed statistically different between embryos with different days of blastocyst formation ([Day 5]: 1.06 vs. [Day 6]: 0.66, p=0.021), and between embryos with different expansion stages ([Expansion 5]: 1.05 vs. [Expansion 6]: 0.49, p=0.012). None or weakly correlated with the maternal age, morphology, ploidy, and gender. Analyzed by the different days of blastocyst formation with fixed expansion score as 5 in the euploid single embryo transfers (eSET), the day 6 eSET showed significantly lower reduced mtDNA ratio (n=139) in failure groups of fetal heartbeat (p=0.004), ongoing pregnancy (p=0.007), and live birth (p=0.01); however, no correlation between mtDNA ratios and pregnancy outcomes was observed in the day 5 eSET (n=1,201). Conclusions: The study first demonstrated that mtDNA ratio was dependent on the days of blastocyst formation while expansion stage was fixed. Lower mtDNA ratios were observed in the day 6 eSET with adverse outcomes. The present stratification analyses reveal that the timeline of embryo is an important covariate to the mtDNA content.

High concordance in preimplantation genetic testing for aneuploidy between automatic identification via Ion S5 and manual identification via Miseq.

The Ion S5 (Thermo Fisher Scientific) and Miseq (Illumina) NGS systems are both widely used in the clinical laboratories conducting PGT-A. Each system employs discrepant library preparation steps, sequencing principles, and data processing algorithms. The automatic interpretation via Ion Reporter software (Thermo Fisher Scientific) and the manual interpretation via BlueFuse Multi software (Illumina) for chromosomal copy number variation (CNV) represent very different reporting approaches. Thus, it is intriguing to compare their ability of ploidy detection as PGT-A/NGS system. In the present study, four aneuploid cell lines were individually mixed with a diploid cell line at different aneuploid ratios of 0% (0:5), 10% (1:9), 20% (1:4), 40% (2:3), 50% (3:3), 60% (3:2), 80% (4:1) and 100% (5:0) to assess the sensitivity and specificity for whole chromosomal and segmental aneuploidy detection. The clinical biopsies of 107 blastocysts from 46 IVF/PGT-A cycles recruited between December 2019 and February 2020 were used to calculate the concordance. Initially, the pre-amplified products were divided into two aliquots for different library preparation procedures of each system. Applying the same calling criteria, automatic identification was achieved through the Ion Reporter, while well-trained technicians manually identified each sample through the BlueFuse Multi. The results displayed that both systems reliably distinguished chromosomal CNV of the mixtures with at least 10% aneuploidy from karyotypically normal samples ([Ion S5] whole-chromosomal duplication: 2.14 vs. 2.05, p value = 0.009, segmental deletion: 1.88 vs. 2.05, p value = 0.003; [Miseq] whole-chromosomal duplication: 2.12 vs. 2.03, p value = 0.047, segmental deletion: 1.82 vs. 2.03, p value = 0.002). The sensitivity and specificity were comparable between the Ion S5 and Miseq ([sensitivity] 93% vs. 90%, p = 0.78; [specificity] 100% vs. 100%, p value = 1.0). In the 107 clinical biopsies, three displayed chaotic patterns (2.8%), which could not be interpreted for the ploidy. The ploidy concordance was 99.04% (103/104) per embryo and 99.47% (2265/2277) per chromosome pair. Since their ability of detection were proven to be similar, the automatic identification in Ion S5 system presents comparatively faster and more standardized performance.

Thermodynamic analysis of remote substrate binding energy in 3α-hydroxysteroid dehydrogenase/carbonyl reductase catalysis.

The binding energy of enzyme and substrate is used to lower the activation energy for the catalytic reaction. 3α-HSD/CR uses remote binding interactions to accelerate the reaction of androsterone with NAD+. Here, we examine the enthalpic and entropic components of the remote binding energy in the 3α-HSD/CR-catalyzed reaction of NAD+ with androsterone versus the substrate analogs, 2-decalol and cyclohexanol, by analyzing the temperature-dependent kinetic parameters through steady-state kinetics. The effects of temperature on kcat/Km for 3α-HSD/CR acting on androsterone, 2-decalol, and cyclohexanol show the reactions are entropically favorable but enthalpically unfavorable. Thermodynamic analysis from the temperature-dependent values of Km and kcat shows the binding of the E-NAD+ complex with either 2-decalol or cyclohexanol to form the ternary complex is endothermic and entropy-driven, and the subsequent conversion to the transition state is both enthalpically and entropically unfavorable. Hence, solvation entropy may play an important role in the binding process through both the desolvation of the solute molecules and the release of bound water molecules from the active site into bulk solvent. As compared to the thermodynamic parameters of 3α-HSD/CR acting on cyclohexanol, the hydrophobic interaction of the B-ring of steroids with the active site of 3α-HSD/CR contributes to catalysis by increasing exclusively the entropy of activation (ΔTΔS‡â€¯= 1.8 kcal/mol), while the BCD-ring of androsterone significantly lowers ΔΔH‡ by 10.4 kcal/mol with a slight entropic penalty of -1.9 kcal/mol. Therefore, the remote non-reacting sites of androsterone may induce a conformational change of the substrate binding loop with an entropic cost for better interaction with the transition state to decrease the enthalpy of activation, significantly increasing catalytic efficiency.

Concordance between different trophectoderm biopsy sites and the inner cell mass of chromosomal composition measured with a next-generation sequencing platform.

STUDY QUESTION: In PGS, does chromosomal constitution differ among trophectoderm (TE) biopsy sites and between them and the inner cell mass (ICM)? SUMMARY ANSWER: The ploidy concordance between ICM and TE was independent of whether the biopsy site in the TE was near to or far from the ICM. WHAT IS KNOWN ALREADY: TE biopsies are considered less harmful to developing embryos than blastomere biopsies. Removal of multi-cellular samples permits high-resolution next-generation sequencing (Veriseq NGS) to detect aneuploidy present in a minority of cells (mosaicism of diploid and aneuploid cells). However, the prevalence of ploidy discrepancies between different TE biopsy sites and the ICM, as well as confined mosaicism (aneuploidy only in a particular area), has not been established. STUDY DESIGN, SIZE, DURATION: Biopsies were taken from a site opposite to the ICM (TE1), near the ICM (TE2) and within the ICM of the same embryo in 33 donated blastocysts obtained from 12 volunteer patients. The samples were analyzed by the Veriseq NGS to assess ploidy concordance. PARTICIPANTS/MATERIALS, SETTING, METHODS: The mean age of the patients was 34.4 years, and samples from all three biopsy sites were achieved in 29 frozen thawed blastocysts. The aneuploid percentage in each sample was interpreted by Veriseq NGS at the finest resolution involving the number of reads after filtering, sample overall noise score, and average quality/alignment scores according to the Veriseq quality control assessment. Ploidy concordance was then assessed between different TE fractions, and between the TE and ICM. MAIN RESULTS AND THE ROLE OF CHANCE: The euploid rates were similar in the TEs and ICM, and no preferential allocation of euploid lineage within a blastocyst was demonstrated. Whether the biopsy site in the TE was near to or far from the ICM, the chromosomal consistency rate was similar [TE1-to-ICM, 86.2% (25/29) versus TE2-to-ICM, 89.7% (26/29); P = 1.0], suggesting that the cells with different chromosomal components may spread randomly throughout the TE. The following two types of inconsistent PGS conclusions between TE and ICM due to confined mosaicism were observed: (i) euploid TE with mosaic ICM (3%) (1/29); and (ii) mosaic TE with euploid ICM (3%) (1/29) or with aneuploid ICM (7%) (2/29). Thus, the overall rate of confined mosaicism was 14% (4/29). LARGE SCALE DATA: N/A. LIMITATION, REASONS FOR CAUTION: The approach used in the present study was affected by biopsy manipulation limitations involving possible cell contamination and the technical challenge of comprehensive chromosomal screening (CCS) procedures. WIDER IMPLICATIONS OF THE FINDINGS: The rate of confined mosaicism in the blastocysts was estimated in this preliminary study, thus, specifying the incidence of biological sampling biases. The results also verified the random distribution of different cell lineages, and the representative value of a single biopsied sample from the TE. STUDY FUNDING AND CONFLICT OF INTEREST(S): No external funding was obtained; all the authors declare no conflicts of interest regarding this study.

Identification of mosaic and segmental aneuploidies by next-generation sequencing in preimplantation genetic screening can improve clinical outcomes compared to array-comparative genomic hybridization.

BACKGROUND: Chromosomal mosaicism is observed as the presence of both euploid and aneuploid cells in a particular blastocyst. Recent studies have reported that the implantation rate of mosaic embryo transfer is remarkably lower than the euploid embryos. The superior capability of next-generation sequencing (NGS) to detect chromosomal mosaicism in preimplantation genetic screening (PGS) remains controversial, and several data displayed similar implantation and pregnancy rates using NGS or array comparative genomic hybridization (aCGH). RESULTS: In this study, the main inconsistency of aneuploidy detection and clinical performance between the NGS and aCGH were assessed. The phase I consisted of a parallel comparison in 182 blastocysts from 45 selected PGS patients for both the NGS and aCGH platforms. The phase II retrospectively compared the clinical outcomes of 90 patients with NGS-screened euploid embryo transfer to that of 129 patients with aCGH-screened euploid embryo transfer. The parallel comparison showed that the inconsistency of embryo euploidy was 11.8% (p = 0.01). Chromosomal mosaicism (10.7% with NGS vs. 3.9% with aCGH) and segmental aneuploidy (10.7% with NGS vs. 6.7% with aCGH) contributed to the discrepancy mainly. The chromosomally mosaic embryos (20%-50% of aneuploidy) and several embryos with segmental aneuploidy (≥10 Mbp) were hard to distinguish using the aCGH platform, but could be clearly identified using the NGS platform. After the first euploid embryo cryotransfer, the ß-HCG(+) rate and implantation rate significantly increased in the PGS/NGS patients (HCG[+] rate: 73.3% in PGS/NGS vs. 60.5% in PGS/aCGH, p = 0.048; implantation rate: 53.2% in PGS/NGS vs. 45.0% in PGS/aCGH, p = 0.043). The clinical and ongoing pregnancy rates appeared higher in the NGS group, but did not reached statistical significance. CONCLUSIONS: The results demonstrated that the NGS platform can identify embryos with chromosomal mosaicism and segmental aneuploidy more precisely than the aCGH platform, and the following clinical performance of NGS was more favorable.

Suitable timing of surgical intervention for ruptured ovarian endometrioma.

OBJECTIVE: Patients with a rare rupture of endometriomas may require surgery. In this retrospective study, we assessed the outcomes of surgical interventions for ruptured ovarian endometriomas. MATERIALS AND METHODS: Forty-three patients who underwent surgical intervention for ruptured ovarian endometriomas were studied. Depending on the latency to surgery and endometrioma recurrence, patients were divided into two groups, and then compared with respect to patient profiles, intraoperative findings, and outcomes. RESULTS: Thirty-one of the 43 patients had a known ovarian endometrioma with an average diameter of 6.04 cm. Seventeen (39.5%) patients had a recurrent ovarian tumor during the postoperative follow up. Patients who underwent surgery within 72 hours or after 72 hours showed no difference in baseline characteristics and most clinical outcomes, except for the choice of surgery (p = 0.003) and future fertility (p = 0.005). CONCLUSION: Comprehensive and early surgical intervention after endometrioma rupture can assist in excluding ovarian malignancy and can reduce the effects of cyst fluids, prevent adhesions, and preserve fertility.

Ultrasound-guided aspiration and ethanol sclerotherapy (EST) for treatment of cyst recurrence in patients after previous endometriosis surgery: analysis of influencing factors using a decision tree.

STUDY OBJECTIVE: To evaluate the efficiency of transvaginal aspiration accompanied by ethanol sclerotherapy for treating cyst recurrence in patients who have previously undergone surgery to treat endometriosis and to analyze various factors that influence success rates using a data mining system. DESIGN: Retrospective cohort study (Canadian Task Force classification II-3). SETTING: Teaching hospital affiliated with Chang Gung University, Taoyuan, Taiwan. PATIENTS: One hundred ninety-six patients with endometrioma recurrence. INTERVENTION: A total of 274 transvaginal aspirations followed by sclerotherapy with 95% ethanol. Treatment times varied from immediate removal (0-10 minutes) to in situ retention. Patients were followed up at 3, 6, and 12 months to detect complications, determine the size and persistence of cysts, obtain the pelvic pain score, and assess for pregnancy or the need for repeat surgical intervention. A decision tree was used to determine factors from the collected data that most influenced the success of treatment. MEASUREMENTS AND MAIN RESULTS: Cyst size was consistently reduced until 6 months after ethanol sclerotherapy. The mean (SD) cyst reduction rate was 37.2% (42.2%), and the pain score reduction rate was 20.5% (71.5%). The antral follicle count was simultaneously increased by 36.4%. Sixty-three patients (23%) required repeated surgery during the observation period and were treated with either repeat aspiration (13.5%) or major laparoscopic or open laparotomic interventions (8.4%). Eighteen of 101 infertile patients (17.8%) achieved pregnancy. The total recovery rate (pregnancy or no persistence of symptoms or cyst) was significantly higher in patients in the groups that received longer treatment (7-10 minutes and retention) than in the groups with shorter treatment (0-6 minute) (47.0% vs 28.7%; p < .005). The highest recovery rate was observed in patients with longer treatment time, smaller cysts (≤5.05 cm), lower CA 125 level (≤62.03 IU/mL), and fewer cysts (≤3 cm) (35 of 49 [71.4%]). In patients with larger cysts and cysts with clear contents, better success can be achieved with longer treatment. The use of postoperative ovarian suppression, traditional Chinese medicine, or no therapy for 6 months before the study was not significant among groups. CONCLUSION: Ultrasound-guided sclerotherapy with 95% ethanol retention is an effective alternative therapy for recurrent ovarian endometrioma, in particular in selected patient groups.

Adnexal torsion in a woman undergoing ovarian hyperstimulation with clomiphene citrate therapy: a case report and review of the literature.

Ovarian stimulation is a unique aid for patients treated for anovulation and an important tool in various assisted reproduction treatments. Clomiphene citrate, an orally active, non-steroidal triphenylethylene derivate, is a commonly prescribed agent for ovulation induction. Clomiphene citrate is considered a safe agent and has rarely been associated with significant side effects. This report describes a case of unilateral adnexal torsion after ovulation induction with clomiphene citrate; we performed unwinding of the adnexum, which appeared ischemic via laparoscopy. Unfortunately, the affected adnexum became hemorrhagic after this approach, which invariably led to its resection.

Long-term follow-up of patients surgically treated for ruptured ovarian endometriotic cysts.

OBJECTIVE: Approximately 4% of women are admitted to hospitals because of ovarian cyst rupture, hemorrhage, or torsion. Endometriotic cyst rupture is a rare surgical emergency associated with severe peritonitis and pelvic adhesion, and we aimed to determine its prognosis and long-term outcome. MATERIALS AND METHODS: We reviewed and analyzed the medical records of 11 patients (mean age, 31.8 ± 7.2 years) with ruptured endometrioma and a history of dysmenorrhea (4.9 ± 2.3 of maximum 10) who were surgically treated, and then regularly followed-up for more than 3 years (range, 35-261 months). RESULTS: Previous ultrasound examinations revealed pelvic cysts in seven patients. Three patients had a history of endometrioma surgery. In the emergency room, eight patients complained of uterine motion tenderness. Sonography revealed residual ovarian tumors (size range, 4.2-10.4 cm), with or without fluid accumulation in the cul-de-sac. Surgical enucleation by laparoscopy or laparotomy revealed high revised American Fertility Society endometriosis scores (78 ± 20.1) as well as high adhesion scores (48.7 ± 11.3). In the postoperative period, four patients had recurrent ovarian tumors that were related to elevated serum cancer antigen 125 levels and high postoperative pain scores. In contrast, three patients who became pregnant during the postoperative period had low serum cancer antigen 125 levels and pain scores. CONCLUSION: Endometrioma rupture should be considered in females presenting with sudden lower abdominal pain, associated with a history of dysmenorrhea and preexisting pelvic cysts. Emergency surgical intervention may lead to a better prognosis, particularly in patients without a history of previous endometrioma surgery.

Prenatal diagnostic rates and postnatal outcomes of fetal orofacial clefts in a Taiwanese population.

OBJECTIVE: To report the incidence, prenatal diagnostic rate, and postnatal outcomes of fetal orofacial cleft at a tertiary referral center in Taiwan. METHODS: The demographic data, maternal and fetal characteristics, and postnatal outcomes for fetuses with cleft lip and/or cleft palate (CL/P) born between January 1998 and December 2008 at Chang Gung Memorial Hospital, Taipei, were reviewed retrospectively, and diagnostic rates were evaluated according to cleft type. RESULTS: Among 26499 deliveries, 84 were affected with CL/P. The mean maternal age and gestational age at detection of CL/P were 30.37 years (range 21-41 years) and 24.7 weeks (range 18-33 weeks), respectively. Thirty-one fetuses had associated structural anomalies, 5 of which involved chromosomal aberrations. CL/P was diagnosed prenatally for 74 (88%) fetuses. After consultations, 17 pregnancies (20%) were aborted. The postnatal survival rate was 95.5% (64/67 infants). The type of cleft had a significant influence on correct prenatal diagnosis (P<0.001). CONCLUSION: For fetuses diagnosed prenatally with an orofacial cleft, access to an experienced craniofacial team, well-planned delivery, and pediatric intensive care led to favorable postnatal outcomes after lethal malformations were excluded through detailed sonographic and chromosomal evaluations.

Effectiveness of ultrasound-guided aspiration and sclerotherapy with 95% ethanol for treatment of recurrent ovarian endometriomas.

OBJECTIVE: To evaluate the effectiveness of transvaginal ultrasound aspiration and ethanol sclerotherapy in patients with recurrent ovarian endometriomas. DESIGN: Retrospective study. SETTING: Teaching hospital affiliated with Chang Gung University, Taipei. PATIENT(S): Patients (n = 108) with recurrent ovarian endometriomas >or=3 cm. INTERVENTION(S): Preoperative evaluation of previous pathology, midcycle serum CA-125 level, and color Doppler ultrasonography to exclude possibility of malignancies. After aspiration, sclerotherapy with 95% ethanol irrigation of the cystic cavity was performed (group 1, n = 78, 0-10 minutes of retention; group 2, n = 30, ethanol left in situ [retention]). MAIN OUTCOME MEASURE(S): Ultrasonography was performed at 3, 6, 9, and 12 months to determine persistence and size of cysts and the number of antral follicles. Pelvic pain score was also determined at those time points. RESULT(S): The 1-year recurrence rate for group 2 patients was significantly lower than for group 1 patients (13.3% vs. 32.1%). Antral follicle count was increased and pain score was decreased in both groups to a similar level. No significant change in CA-125 was observed. CONCLUSION(S): Ultrasound-guided sclerotherapy with 95% ethanol is an effective therapy for ovarian endometriomas. Retention of ethanol is more effective than irrigation only.

Excellent progression-free survival with liposomal doxorubicin for a patient with recurrent ovarian malignant mixed müllerian tumor: case report and literature review.

INTRODUCTION: Ovarian malignant mixed müllerian tumor (MMMT) is a rare, highly aggressive, fatal disease. Patients have a median survival of 18 months and a 5-year survival rate of only 8%. Optimal cytoreduction surgery plus platinum-based combination chemotherapy are associated with better outcomes. CASE REPORT: A 65-year-old patient of stage IIIc ovarian MMMT having obtained a 41-month remission after four courses of aggressive surgical debulking procedures, platinum-containing chemotherapy, and intraoperative radiotherapy suffered from multi-focal recurrences and obtained another 22-month progression-free survival after treatment with monthly liposomal doxorubicin (Lipo-Dox) for 14 courses and Lipo-Dox/carboplatin for subsequent 6 courses without obvious toxicity. DISCUSSION: Liposomal doxorubicin might be useful as salvage chemotherapy for heavily pretreated, recurrent ovarian MMMT. A prospective trial is needed for more proof.