Ultrasound Evaluation of Uterine Cavity Changes After Stimulation for In Vitro Fertilization.

OBJECTIVES: Mock embryo transfer (ET) before in vitro fertilization (IVF) allows for the clinical determination of uterine cavity length (UCL) to optimize embryo placement during clinical ET. Most studies have shown that optimal pregnancy rates occur with clinical ET at a depth of 15 mm from the uterine fundus. In our study, we sought to determine the effect of ovarian stimulation and endometrial preparation on UCL using 2D transabdominal ultrasound. METHODS: We performed a retrospective cohort study comparing documented 2D transabdominal ultrasound measurements of UCL at the time of mock ET and clinical ET. Statistical analyses were performed with SPSS v. 26 with paired sample t-test and significance determined with P < .05. RESULTS: Seventy patients who underwent 91 IVF-ET cycles between 2015 and 2018 at our academic center met inclusion criteria. Patient's demographics include a median age of 34 (interquartile range [IQR]: 31, 37), gravida 1 (IQR: 0, 2), parity 0 (IQR: 0, 0), and body mass index 25.87 (IQR: 21.78, 30.01). There was a statistically significant increase in UCL by 11.9 mm after IVF stimulation (P < .001), compared to mock ET. Mean UCL at the time of mock ET was 7.66 cm (±0.98 cm) and at clinical ET was 8.85 cm (±0.98 cm). CONCLUSIONS: The uterine cavity undergoes a significant length change during ovarian stimulation and endometrial preparation. These findings confirm the remarkable uterine plasticity in response to hormonal stimulation even before pregnancy ensues. These changes in UCL should be considered during ultrasound-guided clinical ET to ensure optimal embryo placement.

Endometrial Abnormalities: Correlation Between Different Diagnostic Modalities.

OBJECTIVES: We sought to evaluate the diagnostic accuracy of transvaginal ultrasound (TVUS) saline infusion sonohysterogram (SIS), and hysteroscopy for diagnosing endometrial abnormalities and their correlation with histological findings. In addition, we sought to validate the subsistence of a more subtle abnormality called "redundant endometrium" (RE). METHODS: Retrospective cohort study of patients presenting with infertility and diagnosed with endometrial abnormalities who underwent hysteroscopy and pathology evaluation. Each patient underwent TVUS at the first visit regardless of the cycle phase, followed by SIS during proliferative phase, and then hysteroscopy, which was performed when abnormal SIS findings were diagnosed. Endometrial abnormalities were categorized as polyps (EP), RE, or normal. Frequencies of the abnormalities were recorded for the 3 imaging modalities and their sensitivity, specificity, positive predictive value, and negative predictive value were calculated for each. RESULTS: A total of 105 TVUS and 73 SIS were performed. Because of the frequent association of EP with RE, when all endometrial pathologies were combined, the three diagnostic modalities showed high sensitivity (TVUS 88.9%, SIS 100%, hysteroscopy 82.9%, respectively), but they also showed low specificity (TVUS 56.7%, SIS 56.1%, hysteroscopy 58.2%, respectively). Pathological evaluation showed isolated RE (26 cases), to harbor polyps (19.2%), disordered endometrium (19.2%), and complex atypical hyperplasia (3.8%). CONCLUSIONS: The three diagnostic modalities showed high sensitivity in diagnosing endometrial abnormalities. We identified RE as an independent endometrial abnormality. Benign endometrium is the predominant histology in RE, however, a small proportion harbors endometrial hyperplasia. Based on these results, we advocate further evaluation when this condition is diagnosed with TVUS, or SIS.

Anti-Müllerian Hormone (AMH) regulates BRCA1 and BRCA2 gene expression after ovarian cortex transplantation.

OBJECTIVE: To test whether recombinant anti-Müllerian hormone (rAMH) could exert an inhibitory function on BRCA1/2 expression in human ovarian cortex. METHODS: Pilot study on ovariectomized nude mice xenotransplanted with human vitrified/warmed ovarian cortex and treated with rAMH via infusion pump. Twelve nude mice were ovariectomized and Alzet pumps delivering 1.23 mcg rAMH/day to reach a serum concentration of 17.5 ng/mL, or placebo (controls), were inserted intraabdominally. Previously vitrified/warmed 2x2 mm ovarian cortex fragments were transplanted on day 7 and then harvested on day 14 after pump placement. PCR analyses determined mRNA levels for BRCA1 and BRCA2 in the human ovarian cortex. RESULTS: In mice treated with rAMH, BRCA1 expression was significantly lower (0.196 fg/µg RNA, IQR 0.158, 0.236) than in controls (0.544 fg/µg RNA, IQR 0.458, 0.554; p = .030), while BRCA2 expression remained similar in rAMH mice (5.355 fg/µg RNA, IQR 4.479, 6.230) and in controls (4.011 fg/µg RNA, IQR 3.650, 4.182; p = .327). CONCLUSION: Administration of rAMH in the peri-transplant period caused downregulation of BRCA1, but not of BRCA2 expression, in human ovarian cortex. These results help our understanding of DNA repair mechanism in the ovarian cortex and identify AMH's possible protective effect on ovarian reserve in BRCA1 mutation carriers.

Pilot study establishing a nomogram of yolk sac growth during the first trimester of pregnancy.

AIM: The yolk sac (YS) has been reported as a reliable predictor of adverse pregnancy outcomes, however, it has always been evaluated cross-sectionally with a single ultrasound per patient. We sought to validate the use of YS dimensions in serial ultrasounds throughout the first 10 weeks of singleton and multiple gestations. METHODS: This was a prospective cohort study where YS diameters were serially obtained with 2D ultrasound in singleton and multiple gestations from 5 to 11 weeks. Nonparametric test were used for comparisons with P < 0.05 indicating significance. RESULTS: One hundred ninety-three patients were included, 42 twins (3 monochorionic and 39 dichorionic), 2 triplets (monochorionic twins plus a singleton) and 148 singleton pregnancies (238 total fetuses). There was no difference in YS dimensions in singleton versus multiple pregnancies. Starting at 5 weeks' gestation, the YS increased 0.4 mm (95% CI 0.3-0.5 mm) per week until 10 weeks' gestation. Forty-five fetuses were lost in the first trimester. The risk of pregnancy loss was higher with a large YS until 8 weeks (P ≤ 0.001), while after 8 weeks it was higher with a small YS (P < 0.005). CONCLUSION: We established a nomogram of YS development during the first 10 weeks of pregnancy. The YS reliably detected pregnancies that ended in loss as early as 6 weeks' gestation. The YS was either smaller or larger than in ongoing pregnancies. While all pregnancies with large YS were lost within 10 weeks, those with smaller YS were lost beyond the first 10 weeks.

Xenotransplantation of pre-pubertal ovarian cortex and prevention of follicle depletion with anti-Müllerian hormone (AMH).

OBJECTIVE: To determine whether recombinant AMH (rAMH) could prevent post-transplant follicular depletion by acting on the stemness markers Oct-4, Sox2, and NANOG. MATERIALS AND METHODS: This was an experimental study where 12 ovariectomized nude mice were xenotransplanted with vitrified/warmed ovarian cortex obtained from a pre-pubertal girl and Alzet pumps delivering rAMH, or placebo (control), were inserted intra-abdominally. Previously vitrified/warmed ovarian cortex fragments were transplanted after 7 days and then harvested after 14 days from pump placement. We performed real-time RT-PCR analyses, ELISA for AMH, FSH, and estradiol, histologic measurement of ovarian follicles, and immunohistochemistry for Ki67 and TUNEL. The main outcome measures were serum levels and tissue expression of the parameters under investigation and follicle count. RESULTS: Serum AMH, FSH, and estradiol reflected post-ovariectomy profiles and were mildly influenced by rAMH administration. Ovarian cortex expression of AMH, AMH-R2, VEGF, GDF9, Oct-4, and Sox2 was lower in rAMH mice than in controls, while NANOG was upregulated. There was a non-significant decrease in primordial follicles after vitrification-warming, and xenotransplantation further decreased this number. There were lower cell replication and depressed apoptosis in the rAMH group. CONCLUSIONS: Administration of recombinant AMH in the peri-transplant period did not protect the initial follicular depletion but decreased apoptosis and cellular activation and regulated stem cell markers' tissue expression. These results aid our understanding of the inhibitory effects of AMH on follicular development and show the benefit of administering exogenous AMH at the time of pre-pubertal ovarian cortex transplant to protect the follicles from pre-activation and premature depletion.

Association between redundant endometrium and endometrial polyps: a pilot study.

BACKGROUND: The aim of this study was to explore the organic features of redundant endometrium (RE), we examined the expression of different endometrial hormone receptors, oncogenes, and cell replication markers, in normal endometrium (NE), endometrial polyps (EP) and RE specimens. METHODS: This was an experimental study examining endometrial tissue expression of estrogen receptors (ER1 and 2), progesterone receptors (PR-A+B), androgen receptor (AR), insulin receptor (Insulin-R), insulin-like growth factor receptor 1 (IGFR-1), thyroid hormone receptor (TH-RB), B-cell lymphoma 2 (Bcl-2), Ki67, HOXA10, in women with NE, EP and RE, of women undergoing hysteroscopy for benign gynecologic pathology. Specimens were separated in 3 groups: NE, EP, RE. Endometrial samples were processed for real-time RT-PCR analyses. Main outcome measure was tissue expression of the markers in the three groups. RESULTS: Of the 16 patients, 2 had NE, 8 had RE, 5 had EP, 1 had both, RE and EP. Compared to NE, RE and EP showed significantly increased Bcl-2, Insulin-R, ER-ß, PR-A+B, and TRB expression (P<0.044), with EP showing significantly increased PR-A+B, compared to RE (3.29±0.47 fg/µg RNA versus 1.86±0.34 fg/µg RNA; P=0.023). The other markers were not significantly different across the three groups: Ki67 appeared non-significantly decreased, while HOXA10, IGF-R1, AR, and ER-α, were non-significantly increased. CONCLUSIONS: RE showed biochemical characteristics different from NE. Similar to endometrial polyps, RE showed enhanced cell differentiation, but not cell replication. These changes in RE could be detrimental for embryo implantation and should be of consideration in women undergoing fertility treatments.

A comparison of four systems for uterine septum diagnosis and indication for surgical correction.

INTRODUCTION: Existing guidelines do not settle on a specific length to indicate surgical incision of subseptations because of differences in the four published diagnostic methods: AFS-10 mm classification, 1988/2003, ESHRE-ESGE classification, 2013, ASRM criteria, 2016- and 5.9-mm length cut-off, 2017. With this review and data analysis we sought to identify the classification method with the most accurate association with early pregnancy loss, as to identify a subseptation length cut-off to indicate surgical correction. EVIDENCE ACQUISITION: We performed an exhaustive literature search of PubMed (MEDLINE), Embase, and Cochrane Library databases until April 20, 2020 (limited to articles published in English) of the terms "uterine septum," "arcuate uterus," "subseptation," "Müllerian anomalies," from 1980-2020. After identifying all the available classifications for uterine subseptations, we performed a secondary data analysis of our departmental database on uterine subseptations and compared the identified classification criteria. Measurement of the subseptation's length was obtained on 2-D and 3-D ultrasound in accordance with the different methods. The incidence of uterine subseptations according to each method's specifications was compared among the groups and the association with pregnancy loss was evaluated. EVIDENCE SYNTHESIS: The database comprised 125 women with uterine subseptations and all four diagnostic systems identified septate uteri within it. The 5.9-mm cut-off diagnosed 89 septate, and 36 normal uteri and was the most inclusive while the ASRM cut-off was the most restrictive one, diagnosing 92/125 as arcuate uteri, only 8/125 as septate, and 25 in the gray zone. The AFS-10 mm criteria diagnosed 92/125 as arcuate, and 33 (26.4%) as septate uteri. Subseptations were inconsistently diagnosed by the ESHRE-ESGE classification, as some subseptations longer than 10 mm would be classified as normal uteri. Five/24 women had had one previous early loss and 19/24 had recurrent pregnancy loss. The 5.9-mm system was the most sensitive, while the ASRM was the least sensitive in predicting pregnancy loss (71.2% vs. 9.5% of septate uteri). CONCLUSIONS: The proposed 5.9-mm cut-off was the most sensitive in diagnosing a septate uterus and in predicting an associated early pregnancy loss. Conversely, the AFS-10 mm and the ASRM were the most restrictive, potentially missing treatment for hazardous subseptations. This update highlights the major weaknesses in the current diagnosis of uterine subseptations and indication for surgical treatment. Standardization of clinical practice is essential for reproductive clinicians and efforts should be made to prevent even one further early pregnancy loss to uterine subseptations.

Fluid deficit calculation at hysteroscopy: could consideration of intraperitoneal fluid accumulation add insight to safety limits?

BACKGROUND: The current literature and guidelines are largely silent regarding the contribution of the fallopian tubes to the fluid deficit (FD) during hysteroscopy. We explored whether the FD could be in part due to transtubal passage. METHODS: This was a prospective cohort study. Patients who underwent hysteroscopy because of benign gynecologic pathology with, or without, laparoscopy were enrolled. The fluid deficit and, in laparoscopic cases, the amount of fluid found in the pelvis were prospectively reported. RESULTS: Comparisons between FD and intraperitoneal fluid were performed. Sixty-five patients were included in the study. Forty-five underwent hysteroscopy prior to laparoscopy and 20 patients underwent hysteroscopy-only. These were further divided into operative hysteroscopy and diagnostic hysteroscopy subgroups. In the laparoscopy group, the average FD was 525.9 mL (95% CI: 482.1-569.7) and the calculated FD due to intravasation was 286.6 mL (95%CI: 253.0-320.3). In the hysteroscopy without laparoscopy group, the average FD was 303.0 mL (95% CI: 85.2-520.8). There was no correlation between the intrauterine fluid pressure and the amount of FD, or the presence of intraperitoneal fluid. CONCLUSIONS: Most women with patent tubes undergoing hysteroscopy have accumulation of distention fluid in the pelvis and that the passage was not correlated with the intrauterine fluid pressure. These findings add new insight to the current guidelines, suggesting more accurate and patient-centered safety protocols.

Early detection of velamentous cord insertion at the eighth week of gestation.

Velamentous cord insertion can be diagnosed at 8 weeks of gestation, earlier than previously reported. Fetal surveillance may be informed and prognosis may be impacted by early diagnosis once viability is reached.

Recombinant Anti-Müllerian Hormone (rAMH) for Stalling In Vitro Granulosa Cell Replication.

To investigate whether recombinant AMH (rAMH) is able to decrease cellular proliferation/apoptosis in luteinized granulosa cells (GCs) through hormonal regulation, a primary culture of GCs was established from GCs obtained at time of oocyte retrieval from follicular fluid of 3 patients. Cells were seeded in well cell culture plates at a density of 100,000 cells/well in medium and treated with rAMH 20 ng/ml (rAMH group), or phosphate-buffered saline (PBS-control group), for 24 h. Total RNA was extracted from all cells, followed by cDNA synthesis and real-time RT-PCR to quantify the expression levels of AMH, AMH-R2, FSH-R, inhibin B, cell proliferation (Ki67), and apoptosis (Caspase 3). We used independent sample t test (SPSS v25) and a p < 0.05 significance. Cellular expressions of AMH, AMH-R2, FSH-R, and inhibin B were reduced greater than 50% in the rAMH group, compared with that of the the control group (p ≤ 0.005 for all). Ki67 and Caspase3 were also reduced greater than 30% in the rAMH group (p ≤ 0.001 for both). Our findings show a direct inhibitory effect of AMH on luteinized GCs' expression of the major regulatory hormones, in addition to a significant decrease in markers of cell proliferation and apoptosis. These results confirm the inhibitory effects of AMH on follicular development.

Restoration of Uterine Cavity Measurements after Surgical Correction.

Objective: We sought to define the uterine and uterine cavity dimensions of subseptate uteri before and after hysteroscopic surgical incision, and compare them to those obtained in normal uteri with 3-D ultrasound. Methods: Two cohorts of consecutive women with normal-appearing uterine cavity and women diagnosed with uterine subseptations, before and after undergoing hysteroscopic incision. 3-D ultrasound was used to measure the uterine cavity width, length, and area on a frozen coronal view of the uterus. Results: A total of 215 women were included: 89 in the normal, and 126 in the subseptate uterus, groups. Uterine length and height were similar in the pre-operative, post-operative subseptate uteri, and in the normal uteri, while the uterine width was significantly greater in the pre-operative (5.1 + 0.8 cm) than post-operative (4.7 + 0.8 cm) and normal uterus (4.6 + 0.7 cm; p < 0.001) groups. The pre-operative uterine cavity length (3.3 + 0.5 cm), width (3.2 + 0.7 cm), and area (4.4 + 1.2 cm2), were significantly greater than the post-operative ones (length 2.9 + 0.4 cm; width 2.6 + 0.6 cm; area 3.7 + 0.8 cm; overall p < 0.001), and became similar to the dimensions of the normal uterus. Of the patients who subsequently conceived, 2.6% miscarried in the corrected subseptation group and 28.8% miscarried in the normal uterus group. Conclusions: We defined the ultrasound dimensions of the uterine cavity in subseptate uteri and their change after surgical correction. Uterine cavity length, width, and area show very little variability in adult normal uteri, while they are increased in uteri with a subseptation greater than 5.9 mm in length, and regain normal measurements after surgical correction.

Early pregnancy ultrasound measurements and prediction of first trimester pregnancy loss: A logistic model.

Our objective was to prospectively validate the use of gestational sac (GS), yolk sac (YS) diameter, crown-rump length (CRL), and embryonal heart rate (HR) dimensions to identify early pregnancy loss. This was a prospective cohort study of first trimester pregnancies. GS and YS diameter, CRL, and HR measurements were serially obtained in singleton and twin pregnancies from 6 through 10 weeks' gestation. Non-parametric tests and logistic regression models were used for comparisons of distributions and testing of associations. A total of 252 patients were included, of which 199 were singleton pregnancies, 51 were twins, and 2 were triplets (304 total fetuses). Fifty-two patients had 61 losses. We built nomograms with the changes of the parameters evaluated in ongoing, as well as in pregnancy loss. In the pregnancies which failed, all the parameters showed significant changes, with different temporal onsets: GS and YS were the first to become abnormal, deviating from normality as early as 6 weeks' gestation (OR 0.01, 95% CI 0.0-0.09, and OR 3.36, 95% CI 1.53-7.34, respectively), followed by changes in HR, and CRL, which became evident at 7 and 8 weeks (OR 0.96, 95% CI 0.92-1.0, and OR 0.59, 95% CI 0.48-0.73, respectively). Our observations showed that, after 5 complete weeks' gestation, a small GS and a large YS reliably predicted pregnancy loss. The YS reliably identified the occurrence of a miscarriage at least 7 days prior its occurrence. CRL and HR became abnormal at a later time in pregnancy and closer to the event. These findings have important implications for patient counseling and care planning, as well as a potential bearing on cost effectiveness within early pregnancy care.

Diagnosis of Placental Position by Early First-Trimester Ultrasound: A Pilot Study.

OBJECTIVE: Conventional wisdom is that placental location cannot be identified before 8 weeks' gestation when the placenta first becomes hyperechogenic on ultrasound. We sought to evaluate whether placental location could be reliably diagnosed between 5 and 6 weeks' gestation. MATERIALS AND METHODS: This was a retrospective analysis of prospectively acquired data. Early placental location was diagnosed by evaluation of the embryonal and yolk sac position inside the gestational sac on transvaginal ultrasound. Placental position was described as anterior, posterior, fundal, or lateral. Early and mid-pregnancy placental locations were compared and coded as being the same, having migrated to an adjacent surface, or being on an opposite surface. RESULTS: A total of 111 patients met study criteria, providing 141 placental locations, comprising 85 singleton and reduced pregnancies and 28 dichorionic twin pregnancies. The most common placental location was anterior in both singleton and twin/triplet pregnancies. Placental location at the mid-pregnancy ultrasound was consistent with early pregnancy location in 100% of cases, with 79.5% (112/141) being on the same surface and 20.5% (29/141) having expanded onto an adjacent surface. Placental location was not associated with pregnancy outcome, although our study may have been underpowered to detect a significant difference. CONCLUSIONS: Placental location diagnosed at 5 to 6 weeks' gestation is consistent with the location on mid-pregnancy ultrasound. Excluding the presence of an ectopic, cornual, or cesarean section scar and uterine subseptation pregnancy in early first trimester would allow a more effective tailoring of pregnancy follow-up.

Anti-Müllerian Hormone (AMH) May Stall Ovarian Cortex Function Through Modulation of Hormone Receptors Other Than the AMH Receptor.

OBJECTIVE: To test whether recombinant anti-Müllerian hormone (AMH) can inhibit ovarian cortex function by modulating the expression of other hormone receptors. MATERIALS AND METHODS: Pilot experimental study with ovarian cortex obtained from 5 patients. Immediately after explant, the ovarian cortex specimens were divided into 5 equal fragments. One fragment was flash-frozen (uncultured) and 4 were incubated for 48 hours at 37°C in a pH-adjusted gamete buffer medium with increasing AMH concentrations of 0, 5, 25, and 50 ng/mL. After incubation, all specimens were rinsed and flash-frozen for polymerase chain reaction (PCR) executed in triplicates. We utilized real-time reverse transcription-polymerase chain reaction (RT-PCR) to determine messenger RNA (mRNA) levels of AMH and its receptor Anti-Müllerian Hormone-Receptor 2 (AMH-R2), follicle stimulating hormone receptor (FSH-R), luteinizing hormone receptor (LH-R), inhibin B, and insulin-like growth factor 1 receptor 1 (IGF1-R1) in ovarian cortex tissue. In addition, we performed Ki-67 immunostaining to evaluate cell proliferation in the treatment groups. RESULTS: Absence of recombinant human AMH (rAMH) caused upregulation of all markers. Exposure to increasing rAMH concentrations caused tissue AMH expression downregulation ( P = .024), while AMH-R2 ( P = .005), FSH-R ( P = .009), LH-R ( P = .003), and inhibin B ( P = .001) mRNA expression followed a bell-shaped response with an increased expression at low dose, followed by a decreased expression at higher doses. Expression of IGF1-R1 was independent ( P = .039) of rAMH exposure. The Ki-67 immunostaining showed an increased cell proliferation in the media control compared to the uncultured and the tissue cultured with rAMH. CONCLUSIONS: Culture with increasing rAMH concentrations caused downregulation of its own, as well as other hormone receptors, and a decreased ovarian cortex cell proliferation. These results help understanding the inhibitory effects of AMH on follicular development.