Human umbilical cord perivascular cells maintain regenerative traits following exposure to cyclophosphamide.

Chemotherapies can cause germ cell depletion and gonadal failure. When injected post-chemotherapy, mesenchymal stromal cells (MSCs) from various sources have been shown to have regenerative effects in rodent models of chemotherapy-induced gonadal injury. Here, we evaluated two properties of a novel source of MSC, first trimester (FTM) human umbilical cord perivascular cells (HUCPVCs) (with increased regenerative potential compared to older sources), that may render them a promising candidate for chemotherapeutic gonadal injury prevention. Firstly, their ability to resist the cytotoxic effects of cyclophosphamide (CTX) in vitro, as compared to term HUCPVCs and bone marrow cells (BMSCs); and secondly, whether they prevent gonadal dysfunction if delivered prior to gonadotoxic therapy in vivo. BMSC, FTM HUCPVC, term HUCPVC, and control NTERA2 cells were treated with moderate (150 µmol/L) and high (300 µmol/L) doses of CTX in vitro. Viability, proliferative capacity, mesenchymal cell lineage markers and differentiation capacity, immunogenicity, and paracrine gene expression were assessed. CTX was administered to Wistar rats 2 days following an intra-ovarian injection of FTM HUCPVC. HUCPVC survival and ovarian follicle numbers were assessed using histological methods. We conclude that FTM HUCPVC maintain key regenerative properties following chemotherapy exposure and that pre-treatment with these cells may prevent CTX-induced ovarian damage in vivo. Therefore, HUCPVCs are promising candidates for fertility preservation.

Human umbilical cord perivascular cells prevent chemotherapeutic drug-induced male infertility in a mouse model.

OBJECTIVE: To study whether intratesticular (IT) administration of 2 sources of human umbilical cord perivascular cells (HUCPVC), rich and potent sources of mesenchymal stromal cells (MSC), before chemotherapy can prevent infertility in a mouse model. DESIGN: Two control groups of CD1 male mice without busulfan (BUS) administration (untreated and IT media injection groups) were included. Experimental groups included IT administration of media, first trimester (FTM) HUCPVCs or term HUCPVCs (n = 5 each) injected 3 days before BUS treatment (20 mg/kg). All groups were included in a mating time course study over 6 months. SETTING: Preclinical study in a fertility center research laboratory. PATIENTS: Not applicable. INTERVENTION: IT delivery of FTM or term HUCPVC before BUS treatment. MAIN OUTCOME MEASURES: Pregnancies, litter sizes, and gross morphology of offspring were monitored. Caudal epididymal sperm concentration, motility, and progressive motility were assessed by computer-assisted sperm analysis. Spermatogenesis was also assessed histologically in testicular tissue sections. RESULTS: FTM and term HUCPVC displayed an MSC-associated immunophenotype and expressed transcripts encoding paracrine factors known to regulate the testicular cell niche. IT administration of FTM and term HUCPVC before chemotherapy promoted the recovery of spermatogenesis and fertility compared with BUS-treated animals that received a media injection. Although the total number of pups sired over 6 months by males treated with FTM or term HUCPVC was reduced compared with untreated or media-injected controls, litter size and sperm parameters in fertile animals did not differ between control and cell-treated groups. CONCLUSION: HUCPVC represent a promising source of MSC-based therapy to prevent gonadotoxic chemotherapeutic drug-induced infertility.

In Vitro Fertilization Success Rates after Surgically Treated Endometriosis and Effect of Time Interval between Surgery and In Vitro Fertilization.

STUDY OBJECTIVE: To evaluate the impact of endometriosis staging and endometriomas on in vitro fertilization (IVF) outcome and to assess the optimal time interval between laparoscopy and IVF. DESIGN: A retrospective clinical study (Canadian Task Force classification II1). SETTING: A university-affiliated private infertility clinic. PATIENTS: Two hundred sixteen infertile patients with endometriosis and 209 infertile patients without endometriosis. INTERVENTIONS: Laparoscopy, IVF. MEASUREMENTS AND MAIN RESULTS: Patients with endometriosis were classified according to American Society for Reproductive Medicine criteria; 58, 67, 63, and 28 patients had stages 1 through 4 disease, respectively. Patients with endometriosis had significantly lower estradiol on trigger day (9986 ± 6710 vs 12 220 ± 9414 pg/mL, respectively) and number of retrieved oocytes (12.7 ± 8.6 vs 14.0 ± 10, respectively) compared with controls. We found a consistent decline in clinical and ongoing pregnancy rates with increasing stage of endometriosis. The presence of endometrioma in patients with stages 3 and 4 endometriosis did not alter IVF outcome. Patients with a time interval of 7 to 12 and 13 to 25 months after surgery had a favorable outcome. CONCLUSION: IVF pregnancy rate was negatively correlated with endometriosis severity. The presence of endometriomas had no impact on IVF clinical outcome. The optimal time to perform IVF appears to be between 7 and 25 months after endometriosis surgery.

Initial germ cell to somatic cell ratio impacts the efficiency of SSC expansion in vitro.

Spermatogonial Stem Cell (SSC) expansion in vitro remains a major challenge in efforts to preserve fertility among pubertal cancer survivor boys. The current study focused on innovative approaches to optimize SSC expansion. Six- to eight-week-old CD-1 murine testicular samples were harvested by mechanical and enzymatic digestion. Cell suspensions were incubated for differential plating (DP). After DP, we established two experiments comparing single vs. repetitive DP (S-DP and R-DP, respectively) until passage 2 (P2) completion. Each experiment included a set of cultures consisting of 5 floating-to-attached cell ratios (5, 10, 15, 20, and 25) and control cultures containing floating cells only. We found similar cell and colony count drops during P0 in both S- and R-DP. During P2, counts increased in S-DP in middle ratios (10, 15, and especially 20) relative to low and high ratios (5 and 25, respectively). Counts dropped extensively in R-DP after passage 2. The superiority of intermediate ratios was demonstrated by enrichment of GFRα1 by qPCR. The optimal ratio of 20 in S-DP contained significantly increased proportions of GFRα1-positive cells (25.8±5.8%) as measured by flow cytometry compared to after DP (1.9±0.7%, p<0.0001), as well as positive immunostaining for GFRα1 and UTF1, with rare Sox9-positive cells. This is the first report of the impact of initial floating-to-attached cell ratios on SSC proliferation in vitro. ABBREVIATIONS: SSC: spermatogonial stem cells; DP: differential plating; NOA: non-obstructive azoospermia; MACS: magnetic-activated cells sorting; FACS: fluorescence-activated cells sorting.

Sperm DNA fragmentation index does not correlate with blastocyst euploidy rate in egg donor cycles.

Sperm DNA fragmentation is a known etiology for male infertility. We evaluated the impact of sperm DNA fragmentation index (DFI) on blastocyst euploidy in IVF cycles with egg donors. This observational retrospective study, which was conducted in a university affiliated fertility clinic, included IVF-ICSI-pre-implantation Genetic Screening (PGS) egg donor cycles in which DFI was tested prior to IVF, between January 1st, 2014 and July 31st, 2016. Twenty-seven cycles with DFI > 15% were included in the study group and compared with 18 cycles of DFI < 15% within control group. Research group participants had significantly lower sperm count and motility (55.4*106/ml and 37.4%, respectively) compared with controls (92.5*106/ml and 55.7%, respectively, p < .05). The groups were similar in terms of donors' demography (age, BMI), ovarian reserve (AMH, AFC) and response to hormonal stimulation (E2 level on triggering day and number of retrieved eggs). Embryo development (from 2PN through day 3 embryos to blastocysts) was similar as well. The number of biopsied blastocysts from study and control groups was 171 and 87, respectively. PGS with array comprehensive genomic hybridization revealed comparable euploidy rates of 69.3% and 67.3%, respectively (p > .05). DFI did not have an impact on the blastocyst euploidy rate in IVF cycles with egg donors.

Significant correlation between anti-müllerian hormone and embryo euploidy in a subpopulation of infertile patients.

Anti-Müllerian hormone (AMH) is a standard marker of ovarian reserve. Correlation between AMH and egg euploidy is controversial. We evaluated the association between AMH and blastocyst euploidy rate examined by pre-implantation genetic screening (PGS). This retrospective study was conducted at the CReATe Fertility Centre. We included single IVF cycles of 216 infertile couples, which resulted in 911 blastocysts subjected to array comparative genomic hybridization and evaluated IVF outcome after embryo transfer. The average age and median AMH of female patients were 37.2 (SD = 3.8) and 20 pmol/l, respectively, and the average euploidy rate was 38.3%. Using multivariate regression controlling for age, antral follicle count, body mass index and parity, there was a significant association between serum AMH and proportion of euploid embryos (P = 0.02), due to the dominant ≤36 age group in which significant correlation between AMH and euploidy rate (P = 0.02) was demonstrated. Clinical outcome was similar, including biochemical, clinical and ongoing pregnancy rates as well as pregnancy loss. This study shows a correlation between AMH and aneuploidy rate, specifically among infertile patients younger than 37 years old. Study limitations are discussed.

Sperm DNA fragmentation index does not correlate with blastocyst aneuploidy or morphological grading.

High DNA fragmentation index (DFI) may be associated with poor outcome after IVF. Our aim was to determine whether DFI impacts blastocyst quality or clinical outcome. This retrospective study included 134 couples who underwent 177 IVF-ICSI and pre-implantation genetic screening (PGS) cycles during January 1st, 2014-March 31st, 2016 and had documented previous DFI. Group 1 (DFI>30%) encompassed 25 couples who underwent 36 cycles; Group 2 (DFI 15-30%) included 45 couples and 57 cycles; group 3 (DFI<15%) included 64 couples and 83 cycles. Male partners within group 1 were older (45.1 compared to 40.6 and 38.3 years, respectively, p<0.05), had higher BMI (32.4 compared to 26.6 and 25.8 respectively, p<0.05) and lower sperm count and motility (46*106/ml and 35.5%, respectively) compared to groups 2 (61.8*106/ml and 46.6%, respectively) and 3 (75.8*106/ml and 55.1%, respectively, p<0.05). Female parameters including ovarian reserve and response and embryo development were similar. Total numbers of biopsied blastocysts were 116, 175 and 259 in groups 1, 2 and 3, respectively. PGS for 24 chromosomes revealed comparable euploidy rate of 46-50.4%, with a similar morphological classification. No significant differences were found regarding pregnancy rates or pregnancy loss. It seems that DFI doesn't correlate with blastocyst aneuploidy or morphological grading.

Optimal culture conditions are critical for efficient expansion of human testicular somatic and germ cells in vitro.

OBJECTIVE: To optimize culture conditions for human testicular somatic cells (TSCs) and spermatogonial stem cells. DESIGN: Basic science study. SETTING: Urology clinic and stem cell research laboratory. PATIENT(S): Eight human testicular samples. INTERVENTIONS(S): Testicular tissues were processed by mechanical and enzymatic digestion. Cell suspensions were subjected to differential plating (DP) after which floating cells (representing germ cells) were removed and attached cells (representing TSCs) were cultured for 2 passages (P0-P1) in StemPro-34- or DMEM-F12-based medium. Germ cell cultures were established in both media for 12 days. MAIN OUTCOME MEASURE(S): TSC cultures: proliferation doubling time (PDT), fluorescence-activated cell sorting for CD90, next-generation sequencing for 89 RNA transcripts, immunocytochemistry for TSC and germ cell markers, and conditioned media analysis; germ cell cultures: number of aggregates. RESULT(S): TSCs had significantly prolonged PDT in DMEM-F12 versus StemPro-34 (319.6 ± 275.8 h and 110.5 ± 68.3 h, respectively). The proportion of CD90-positive cells increased after P1 in StemPro-34 and DMEM-F12 (90.1 ± 10.8% and 76.5 ± 17.4%, respectively) versus after DP (66.3 ± 7%). Samples from both media after P1 clustered closely in the principle components analysis map whereas those after DP did not. After P1 in either medium, CD90-positive cells expressed TSC markers only, and fibroblast growth factor 2 and bone morphogenetic protein 4 were detected in conditioned medium. A higher number of germ cell aggregates formed in DMEM-F12 (59 ± 39 vs. 28 ± 17, respectively). CONCLUSION(S): Use of DMEM-F12 reduces TSC proliferation while preserving their unique characteristics, leading to improved germ cell aggregates formation compared with StemPro-34, the standard basal medium used in the majority of previous reports.

Lower complication rates with office-based D&C under ultrasound guidance for missed abortion.

BACKGROUND: Missed abortion (MA) can be managed expectantly, medically or surgically. Surgical management has been performed safely in the office setting by suction dilation and curettage (D&C). Prior studies suggest that intraoperative ultrasound guidance (USG) may reduce complications for first-trimester therapeutic abortion. The aim of this study was to evaluate the safety of office D&C for MA using real-time USG. METHODS: This retrospective cohort study included 255 patients who underwent office D&C under USG for first trimester MA at a single university-affiliated fertility clinic during January 2011-December 2013. Transabdominal USG was utilized during the procedure and was immediately followed by a transvaginal ultrasound examination to confirm full evacuation. Intra- and postoperative complication rates were compared to previously published data. RESULTS: There were no intraoperative complications, including excessive blood loss or uterine perforation. Two of the 255 patients (0.87%) were diagnosed with RPOCs requiring uterine re-evacuation. This rate of RPOCs was superior to rates previously reported for D&Cs without USG (2.6-4.9%, P=0.046). There were no other post procedure complications identified. CONCLUSIONS: We observed very low complications rate in Office-based D&C under USG, lower than those reported in the literature with unguided D&C.

Recurrent implantation failure: a comprehensive review.

Successful pregnancy involves a synchronized, coordinated cross-talk between an embryo capable of implanting, and an endometrium enabling implantation. Recurrent implantation failure (RIF) refers to unsuccessful implantation after repeated transfers of morphologically good quality embryos into a normal uterus. The etiology for RIF can be attributed to the embryo itself, the mother or, in some cases, both. Despite extensive research on underlying causes for RIF, our understanding of this condition is still limited. With the evolving molecular technologies, efforts are focused on studying the implantation process itself, including the molecular aspects of endometrial-embryonic interactions, normal human embryonic development, and preimplantation genetic evaluation. This knowledge will pave the way toward new diagnostic and therapeutic strategies for RIF. In this article, we present a comprehensive review of our current knowledge on this topic.

CD9 is expressed on human male germ cells that have a long-term repopulation potential after transplantation into mouse testes.

Human spermatogonial stem cells (SSCs) play critical roles in lifelong maintenance of male fertility and regeneration of spermatogenesis. These cells are expected to provide an important resource for male fertility preservation and restoration. A basic strategy has been proposed that would involve harvesting testis biopsy specimens from a cancer patient prior to cancer therapies, and transplanting them back to the patient at a later time; then, SSCs included in the specimens would regenerate spermatogenesis. To clinically apply this strategy, isolating live human SSCs is important. In this study, we investigated whether CD9, a known rodent SSC marker, is expressed on human male germ cells that can repopulate recipient mouse testes upon transplantation. Testicular tissues were obtained from men with obstructive azoospermia. Using immunohistochemistry, we found that CD9 was expressed in human male germ cells in the basal compartment of the seminiferous epithelium. Following immunomagnetic cell sorting, CD9-positive cells were enriched for germ cells expressing MAGEA4, which is expressed by spermatogonia and some early spermatocytes, compared with unsorted cells. We then transplanted CD9-positive cells into nude mouse testes and detected an approximately 3- to 4-fold enrichment of human germ cells that repopulated mouse testes for at least 4 mo after transplantation, compared with unsorted cells. We also observed that some cell turnover occurred in human germ cell colonies in recipient testes. These results demonstrate that CD9 identifies human male germ cells with capability of long-term survival and cell turnover in the xenogeneic testis environment.