Guidelines for the management of Helicobacter pylori infection in Japan: 2016 Revised Edition.

BACKGROUND: Since "Helicobacter pylori (H. pylori) infection" was set as the indication in the Japanese Society for Helicobacter Research (JSHR) Guidelines 2009, eradication treatment for H. pylori gastritis is covered under insurance since 2013 in Japan, and the number of H. pylori eradication has rapidly increased. Under such circumstances, JSHR has made the third revision to the "Guidelines for diagnosis and treatment of H. pylori infection" for the first time in 7 years. METHODS: The Guideline Committee held 10 meetings. Articles published between the establishment of the 2009 Guidelines and March 2016 were reviewed and classified according to the evidence level; the statements were revised on the basis of this review. After inviting public comments, the revised statements were finalized using the Delphi method. RESULTS: There was no change in the basic policy that H. pylori infectious disease is an indication for eradication. Other diseases presumed to be associated with H. pylori infection were added as indications. Serum pepsinogen level, endoscopic examination, and X-ray examination were added to the diagnostic methods. The effects of 1-week triple therapy consisting of potassium-competitive acid blocker (P-CAB), amoxicillin, and clarithromycin have improved, and high eradication rates can also be expected with proton pump inhibitors (PPI) or P-CAB combined with amoxicillin and metronidazole. If the susceptibility test is not performed, the triple PPI or P-CAB/amoxicillin/metronidazole therapy should be chosen, because the PPI/amoxicillin/metronidazole combination demonstrated a significantly higher eradication rate than PPI/amoxicillin/clarithromycin. In the proposal for gastric cancer prevention, we divided gastric cancer prevention measures by age from adolescent to elderly, who are at an increased gastric cancer risk, and presented measures for gastric cancer prevention primarily based on H. pylori eradication. CONCLUSION: We expect the revised guidelines to facilitate appropriate interventions for patients with H. pylori infection and accomplish its eradication and prevention of gastric cancer.

Vonoprazan-based triple therapy is non-inferior to susceptibility-guided proton pump inhibitor-based triple therapy for Helicobacter pylori eradication.

BACKGROUND: All Helicobacter pylori-infected patients are recommended for eradication with an appropriate regimen in each geographic area. The choice of the therapy is somewhat dependent on the antimicrobial susceptibility. The rate of clarithromycin resistance has been increasing and is associated with failure; thus, susceptibility testing is recommended before triple therapy with clarithromycin. However, antimicrobial susceptibility testing is not yet clinically available and an alternative newly developed acid inhibitor vonoprazan is used for triple therapy in Japan. The aim of this study was to determine whether vonoprazan-based triple therapy is plausible treatment in H. pylori eradication. METHODS: A retrospective observational study of H. pylori eradication was conducted in a single institute. The patients who requested antimicrobial susceptibility testing were treated with susceptibility-guided proton pump inhibitor-based triple therapy in International University of Health and Welfare Hospital from 2013 to 2016. Other patients were treated with empirical treatment with a proton pump inhibitor. From 2015 to 2016, vonoprazan-based triple treatment (vonoprazan, 20 mg; amoxicillin, 750 mg; and clarithromycin, 200 or 400 mg, b.i.d.) was conducted, and its effectiveness was compared with susceptibility-guided proton pump inhibitor-based triple therapy. We also investigated the improvement in eradication rate when antimicrobial susceptibility testing was performed, and compared the outcomes of vonoprazan-based and proton pump inhibitor-based empirical therapy. RESULTS: A total of 1355 patients who received first-line eradication treatment were enrolled in the present study. The eradication rates of the empirical proton pump inhibitor-based therapy and the vonoprazan-based therapy group in a per-protocol analysis were 86.3% (95% CI 83.8-88.8) and 97.4% (95% CI 95.7-99.1), respectively. In 212 patients who received antimicrobial susceptibility testing, the rate of clarithromycin resistant was 23.5% and the eradication rate in susceptibility-guided treatment was 95.7% (95% CI 92.9-98.4). The difference between susceptibility-guided and vonoprazan-based therapy was - 1.7% (95% CI - 4.9 to 1.5%), and the non-inferiority of vonoprazan-based triple therapy was confirmed. CONCLUSIONS: Vonoprazan-based triple therapy was effective as susceptibility-guided triple therapy for H. pylori eradication. An empirical triple therapy with vonoprazan is preferable even in area with high rates of clarithromycin-resistance. Trial registration The study was retrospectively registered in University Hospital Medical Information Network (UMIN000032351).

[Diagnosis and treatment of aspirin--and NSAID-induced peptic ulcers].

A Japanese case-control study showed the odds ratio of upper gastrointestinal bleeding was 5.5 for aspirin and 6.1 for other NSAIDs. A Japanese cohort study showed that peptic ulcers were found in 6.5% of 1,454 patients receiving low-dose aspirin (LDA). Some endoscopic studies reported that NSAID users often had antral, multiple, and irregular ulcers, irrespective of Helicobacter pylori status. Proton-pump inhibitor (PPI) and misoprostol should be used for therapy for NSAID-ulcers. Maintenance therapy with PPI should be given for prevention of relapse of ulcers of NSAIDs and LDA users. PPI and histamine 2-recetor antagonist were effective for prevention of upper GI mucosal injury in patients receiving LDA.

Clinical management of Helicobacter pylori--the Japanese perspective.

BACKGROUND: After the approval of health insurance coverage of eradication therapy for Helicobacter pylori-positive peptic ulcer disease (PUD) in 2000, comprehensive coverage for H. pylori infection itself was implemented in 2013. METHODS: We did a literature search using PubMed database on the management of H. pylori infection including indications, regimens, outcomes of current eradication therapies, trends of antibiotic resistance rates and proposed third-line rescue therapy in Japan. We also collected data on changes of eradication rates in our hospital by searching electronic medical records. RESULTS: After implementation of insurance coverage of eradication therapy for PUD, dramatic reduction of the number patients with PUD as well as spending on ulcer drug was documented. According to the current regulation, proton pump inhibitor (PPI)-based triple therapy with 2 antibiotics, amoxicillin (AMPC) plus clarithromycin, for 7 days is approved as the first-line therapy. After failure of the first-line therapy, PPI plus AMPC and metronidazole is authorized as the second line, which maintains an excellent eradication rate of over 90% in Japan. When these two therapies fail, a sitafloxacin-based therapy seems to be most promising among many rescue regimens. CONCLUSION: Comprehensive public health insurance coverage of H. pylori infection will promote eradication in Japanese people infected with H. pylori, whose risk of developing gastric cancer has been shown to be high. It also provides us a unique opportunity to study whether the broader indications can accelerate the reduction of gastric cancer in Japan in the same way we witnessed the reduction of PUD.

[Therapy for prevention of upper gastrointestinal bleeding in patients receiving anti-thrombotic therapy].

Meta-analysis of randomized controlled studies and case-control studies showed that the risk of low-dose aspirin for upper gastrointestinal bleeding was 1.6-8.2. Some reports showed that the risk of upper gastrointestinal bleeding (UGIB) was high in patients receiving ticlopidine or clopidogrel. A meta-analysis study reported that data regarding the risk for GI bleeding of new direct oral anticoagulants (apixaban, dabigatran and rivaroxaban) were inconclusive. Proton-pump inhibitor and histamine 2-recetor antagonist were effective for prevention of upper GI mucosal injury in patients receiving low-dose aspirin. Some case-control studies showed that PPI use significantly reduced the risk of UGIB in patients with receiving clopidogrel, but not in those with receiving warfarin.

Diagnosis of depressed-type early gastric cancer using small-caliber endoscopy with flexible spectral imaging color enhancement.

BACKGROUND AND AIM: Small-caliber endoscopy has lower resolution than normal-caliber endoscopy, limiting its use in routine outpatient practice. Flexible spectral imaging color enhancement (FICE) strengthens the color contrast of depressed-type early gastric cancer without magnification. The aim of the present study was to evaluate the detection of depressed-type early gastric cancer using small-caliber endoscopy with the FICE system. METHODS: Eighty-two patients diagnosed with depressed-type early gastric cancer by standard endoscopy and biopsy were evaluated by small-caliber endoscopy. FICE images and conventional images were compared. Color differences in all 82 lesions were measured between malignant lesions and the surrounding mucosa using the Commission Internationale de L'Eclairage (CIE) 1976 color space. RESULTS: Most cancers were readily detected as reddish lesions on FICE images. Lines of demarcation between the malignant lesion and the surrounding mucosa were easily identified with FICE images, as such cancers could be clearly distinguished from the surrounding atrophic mucosa. Greater median color differences between malignant lesions and the surrounding mucosa were present in FICE images compared with conventional images, resulting in images with better contrast (27.2 vs 18.7, P<0.0001). CONCLUSIONS: Small-caliber endoscopy with the FICE system provides better color contrast of depressed-type early gastric cancers than conventional small-caliber endoscopy, and the FICE system may facilitate the diagnosis of this type of cancer as a new endoscopic modality.

Evidence-based clinical practice guidelines for peptic ulcer disease 2020.

The Japanese Society of Gastroenterology (JSGE) revised the third edition of evidence-based clinical practice guidelines for peptic ulcer disease in 2020 and created an English version. The revised guidelines consist of nine items: epidemiology, hemorrhagic gastric and duodenal ulcers, Helicobacter pylori (H. pylori) eradication therapy, non-eradication therapy, drug-induced ulcers, non-H. pylori, and nonsteroidal anti-inflammatory drug (NSAID) ulcers, remnant gastric ulcers, surgical treatment, and conservative therapy for perforation and stenosis. Therapeutic algorithms for the treatment of peptic ulcers differ based on ulcer complications. In patients with NSAID-induced ulcers, NSAIDs are discontinued and anti-ulcer therapy is administered. If NSAIDs cannot be discontinued, the ulcer is treated with proton pump inhibitors (PPIs). Vonoprazan (VPZ) with antibiotics is recommended as the first-line treatment for H. pylori eradication, and PPIs or VPZ with antibiotics is recommended as a second-line therapy. Patients who do not use NSAIDs and are H. pylori negative are considered to have idiopathic peptic ulcers. Algorithms for the prevention of NSAID- and low-dose aspirin (LDA)-related ulcers are presented in this guideline. These algorithms differ based on the concomitant use of LDA or NSAIDs and ulcer history or hemorrhagic ulcer history. In patients with a history of ulcers receiving NSAID therapy, PPIs with or without celecoxib are recommended and the administration of VPZ is suggested for the prevention of ulcer recurrence. In patients with a history of ulcers receiving LDA therapy, PPIs or VPZ are recommended and the administration of a histamine 2-receptor antagonist is suggested for the prevention of ulcer recurrence.

Guidelines for the management of Helicobacter pylori infection in Japan: 2009 revised edition.

BACKGROUND: Over the past few years, the profile of Helicobacter pylori infection has changed in Japan. In particular, the relationship between H. pylori and gastric cancer has been demonstrated more clearly. Accordingly, the committee of the Japanese Society for Helicobacter Research has revised the guidelines for diagnosis and treatment of H. pylori infection in Japan. MATERIALS AND METHODS: Four meetings of guidelines preparation committee were held from July 2007 to December 2008. In the new guidelines, recommendations for treatment have been classified into five grades according to the Minds Recommendation Grades, while the level of evidence has been classified into six grades. The Japanese national health insurance system was not taken into consideration when preparing these guidelines. RESULTS: Helicobacter pylori eradication therapy achieved a Grade A recommendation, being useful for the treatment of gastric or duodenal ulcer, for the treatment and prevention of H. pylori-associated diseases such as gastric cancer, and for inhibiting the spread of H. pylori infection. Levels of evidence were determined for each disease associated with H. pylori infection. For the diagnosis of H. pylori infection, measurement of H. pylori antigen in the feces was added to the tests not requiring biopsy. One week of proton-pump inhibitor-based triple therapy (including amoxicillin and metronidazole) was recommended as second-line therapy after failure of first-line eradication therapy. CONCLUSION: The revised Japanese guidelines for H. pylori are based on scientific evidence and avoid the administrative restraints that applied to earlier versions.

[The present state and problem of gastric ulcer caused by low-dose aspirin].

Upper gastrointestinal (GI) injuries induced by non-steroidal anti-inflammatory drugs (NSAID) and low-dose aspirin (LDA) have been increasing, because the number of patients who need to use NSAID, LDA, other anti-platelet drugs and anti-coagulants have been increasing. The aging is one of the most important risk factors of upper GI injuries induced by LDA, such as gastric ulcer. Since atypical symptoms often lead to a delay in diagnosis and treatment in the elderly patients, endoscopic examination should be considered especially in the elderly patients to detect upper GI lesions before using LDA.

[Treatment and prevention of gastric ulcers in elderly persons].

Treatment and prevention of gastric ulcers is not different between young and elderly patients. Eradication therapy for H. pylori infection should be made for elderly patients with H. pylori-gastric ulcers, because the incidence of adverse events is not higher than in young patients. NSAIDs are often given elderly persons, and aging is a risk factor of the development of NSAID-related ulcer. If elderly patients have some risk factors (high dose NSAID therapy, past history of uncomplicated ulcers or concurrent use of aspirin, corticosteroids or anticoagulants), prophylactic therapy for NSAID-ulcers should be made.

Clarithromycin Versus Metronidazole in First-Line Helicobacter Pylori Triple Eradication Therapy Based on Resistance to Antimicrobial Agents: Meta-Analysis.

BACKGROUND: International treatment guidelines for Helicobacter pylori infection recommend a proton pump inhibitor (PPI)/amoxicillin/clarithromycin (CAM) regimen (PAC) or PPI/amoxicillin/metronidazole (MNZ) regimen (PAM) as first-line therapy based on culture and sensitivity testing. As incidence rates of antimicrobial agent-resistant strains are changing year by year, it is important to reevaluate the efficacy of eradication regimens. We performed a meta-analysis to evaluate the efficacy and safety of PAC and PAM based on different locations categorized by the reported incidence of CAM- and MNZ-resistant strains. METHODS: Randomized control trials (RCTs) comparing eradication rates between PAC and PAM first-line treatment up to December 2018 were included. We divided RCTs into four groups based on resistance to CAM (< 15% or ≥ 15%) and MNZ (< 15% or ≥ 15%). RESULTS: A total of 27 studies (4825 patients) were included. Overall eradication rates between PAC and PAM were similar (74.8% and 72.5%, relative risk (RR): 1.13, 95% confidence interval (CI): 0.91-1.39, P = 0.27) in the intention-to-treat analysis. In areas with low MNZ- and high CAM-resistance rates, PAM had a significantly higher eradication rate than PAC (92.5% vs. 70.8%, RR: 0.29, 95% CI: 0.13-0.68). In areas with high MNZ- and low CAM-resistance rates, the eradication rate with PAC was only 72.9%. CONCLUSIONS: Overall eradication rates with PAC and PAM were equivalent worldwide. In low MNZ-resistance areas, PAM may be recommended as first-line therapy. However, the efficacy of PAC may be insufficient, irrespective of susceptibility to CAM.

Diagnosis of elevated-type early gastric cancers by the optimal band imaging system.

BACKGROUND: The endoscopic diagnosis of an elevated-type early gastric cancer is often difficult. The optimal band imaging (OBI) system can reconstruct the best spectral images decomposed from ordinary endoscopic images with free selection of 3 wavelengths and provide unmagnified images with high light intensity as well as magnified images. OBJECTIVE: To evaluate whether the OBI system facilitates detection of the demarcation lines between an elevated-type early gastric cancer and surrounding tissue and thus is more helpful for performing endoscopic therapy. DESIGN: A prospective study. SETTING: Jichi Medical University, Japan. PATIENTS: Seventy-five patients, 81 lesions with an elevated-type early gastric cancer. MAIN OUTCOME MEASUREMENTS: A comparison between OBI images and conventional endoscopic images in the identification of the demarcation lines of an elevated-type early gastric cancer without magnification and the rate of success in identifying the abnormal surface structure of cancer by using low-magnified OBI images. RESULTS: Demarcation lines were easily identified in OBI images, even without magnification, because such cancers could be clearly distinguished from the surrounding whitish atrophic mucosa. Inexperienced endoscopists could determine demarcation lines with significantly greater accuracy with unmagnified OBI images than with conventional images. With 40-fold magnification, irregular microstructural or nonstructural patterns were also found within cancer lesions in all cases studied but in none of the cases in the surrounding noncancerous mucosa. CONCLUSIONS: The new contrast images obtained with the OBI system enable better determination of the demarcation lines of elevated-type early gastric cancers, and this system may be useful for performing endoscopic therapy of this type of cancer as a new endoscopic modality.

[Second-line therapy for patients with Helicobacter pylori eradication failure].

Meta-analyses showed that quadruple therapy, ranitidine bismuth-based triple therapy and levofloxacin-based triple therapy were the most effective for the second-line H. pylori eradication therapy. In Japan, many studies showed that triple therapy with proton pump inhibitor (PPI), amoxicillin (AMPC) and metronidazole was effective after eradication failure using triple therapy with PPI, AMPC and clarithromycin. The intention-to-treat eradication rates were 81-97%, and the incidence rates of adverse events were 8-33%.

[Helicobacter pylori eradication therapy for peptic ulcer disease].

Meta-analyses showed that Helicobacter pylori eradication therapy was effective for healing and prevention of recurrence of peptic ulcers in H. pylori-positive patients and that treatment of H. pylori infection was more effective than antisecretory non-eradicating therapy (with or without long-term maintenance antisecretory therapy) in preventing recurrent bleeding from peptic ulcer. H. pylori eradication therapy is effective for prevention of development of NSAID-ulcers in NSAID-naïve patients. For prevention of recurrence of NSAID-ulcers, therapy with proton pump inhibitor is effective.

Assessment of atrophic gastritis using the OLGA system.

BACKGROUND: An international group of gastroenterologists and pathologists (Operative Link for Gastritis Assessment (OLGA)) proposed the staging system of atrophy. The aim of this study was to assess the severity of atrophic gastritis using the OLGA system. MATERIALS AND METHODS: The subjects comprised 163 H. pylori-positive patients: 18 with early gastric cancers of the intestinal type (GC), 55 with atrophic gastritis (AG), 49 with gastric ulcers or scars (GU), and 41 with duodenal ulcers or scars (DU). Biopsies were taken from the lesser and greater curvatures of the antrum and middle body. The OLGA gastritis stage (0-IV) (the severity and topography of atrophy) was obtained by combining antral with body atrophy scores. The gastritis grade (the severity and topography of inflammation) was obtained by combining antral and body inflammation scores. RESULTS: Most (84%) of patients with GC showed stage III or IV. Gastritis stages were significantly higher in patients with GC than in those with AG, GU, and DU. Gastritis stage became higher with age. Gastritis grades were slightly higher in patients with AG than in others. CONCLUSIONS: Our results indicate that higher stages are found in patients with GC using the OLGA staging system and that the high risk of GC can be recognized. It is simple to use and useful for the assessment of the severity of atrophic gastritis.

A close relationship between intestinal metaplasia and Cdx2 expression in human gallbladders with cholelithiasis.

We previously reported a case of a human gallbladder with cholelithiasis consisting of intestinal metaplasia with the expression of caudal-related homeobox transcription factor (Cdx2). However, it is unclear how often intestinal metaplasia and Cdx2 expression occur in human, nontumorous gallbladders with cholelithiasis. We studied the incidence of intestinal metaplasia and Cdx2 expression in human gallbladders with cholelithiasis. Gallbladders were resected under laparoscopy from 103 patients with cholelithiasis between September 2003 and March 2005. The mean age of the patients was 59.6 +/- 15.0 years (range, 22-92 years). We retrospectively reviewed these cases to look for the presence of intestinal metaplasia and the expression of Cdx2. In addition, the characteristics of intestinal metaplasia were examined by immunostaining for Muc2, chromogranin A, and serotonin. Intestinal metaplasia was found in 11.7% (12/103) of the gallbladders with cholelithiasis. The mean ages of patients with and without intestinal metaplasia were 60.8 +/- 15.4 and 59.4 +/- 14.9 years, respectively. Cdx2, Muc2, chromogranin A, and serotonin were expressed in 91.7% (11/12), 91.7% (11/12), 83.3% (10/12), and 50.0% (6/12) in intestinal metaplastic mucosa, respectively. Only one case (1.1%) that expressed Cdx2 without intestinal metaplasia did not express Muc2, chromogranin A, and serotonin. We found that 10.7% (11/103) of nontumorous gallbladders resected because of cholelithiasis under laparoscopy revealed intestinal metaplasia with Cdx2 expression.

Changes in plasma ghrelin levels, gastric ghrelin production, and body weight after Helicobacter pylori cure.

BACKGROUND: Ghrelin is a body weight-regulating peptide produced and secreted primarily by the gastric mucosa. Helicobacter pylori infection impairs gastric ghrelin production, leading to a lower plasma ghrelin concentration. However, the effect of H. pylori eradication on plasma ghrelin levels and its relation to body weight change after H. pylori cure are still uncertain. We examined the association of plasma ghrelin levels with gastric ghrelin production and body weight change before and after H. pylori eradication. METHODS: Plasma ghrelin concentrations, gastric ghrelin expression, and body weight were determined in a total of 134 consecutive individuals before and 12 weeks after successful H. pylori eradication. Gastric ghrelin expression was evaluated by determining mRNA expression levels and the number of ghrelin-producing cells in gastric mucosa biopsy specimens by real-time reverse transcriptase-polymerase chain reaction and immunohistochemistry, respectively. RESULTS: Plasma ghrelin concentration increased in 50 patients and decreased in 84 patients after H. pylori eradication. After H. pylori cure, however, gastric preproghrelin mRNA expression was increased nearly fourfold (P < 0.0001), and the number of ghrelin-positive cells was increased or unchanged. In contrast, plasma ghrelin changes after H. pylori cure were inversely correlated with both body weight change (P < 0.0001) and initial plasma ghrelin levels (P < 0.0001). CONCLUSIONS: Changes in plasma ghrelin concentrations before and after H. pylori cure were inversely correlated with body weight change and initial plasma ghrelin levels but not with gastric ghrelin production in Japanese patients.

Development of gastric carcinoma from intestinal metaplasia in Cdx2-transgenic mice.

In the progression of chronic gastritis, gastric mucosal cells deviate from the normal pathway of gastric differentiation to an intestinal phenotype. Many epidemiologic studies have found an association between the formation of intestinal metaplasia and the development of gastric carcinoma. However, there is no direct evidence that shows intestinal metaplasia is a precursor lesion of gastric carcinoma, to date. We periodically examined the intestinal metaplastic mucosa of Cdx2-transgenic mice we have previously generated. Gastric polyps developed from intestinal metaplastic mucosa in all stomachs of Cdx2-transgenic mice examined. These gastric polyps consisted of intestinal-type adenocarcinoma that invaded the submucosa and muscularis propria and occasionally spread into the subserosa. p53 and APC gene mutations were recognized in the adenocarcinomas. The participation of APC and p53 gene mutations in gastric carcinogenesis from the intestinal metaplasia was verified by the Cdx2-transgenic mice, carrying Apc(Min) mutation or p53 deficiency, that developed gastric polyps much earlier than Cdx2 alone. We successfully showed that long-term intestinal metaplasia induces invasive gastric carcinoma. These results indicate that intestinal metaplasia itself plays a significant role in the genesis and progression of gastric carcinoma.

Cdx2 specifies the differentiation of morphological as well as functional absorptive enterocytes of the small intestine.

Many transcription factors are involved in the molecular control of intestinal epithelial cell differentiation. We report in this study that the transcription factor Cdx2 functions to define absorptive enterocytes during intestinal epithelial differentiation. Cdx2 is expressed in the villi of the normal small intestine. Intestinal metaplasia, which expresses Cdx2, occurs as a pathological condition in gastric mucosa. We have previously established Cdx2 transgenic mice expressing Cdx2 exclusively in the gastric epithelium. In this study using Cdx2 transgenic mice, we show that Cdx2 plays a key role in the differentiation of intestinal absorptive enterocytes. The gastric mucosa of Cdx2 transgenic mice was morphologically completely changed into intestinal metaplastic mucosa. Absorptive enterocytes had microvilli which were observed by electron microscope. The intestinal metaplastic mucosa of Cdx2 transgenic mice expressed sucrase and peptide transporter PepT1. Disaccharidase and leucine aminopeptidase activities were observed in the intestinal metaplastic mucosa. Glucose and amino acids were absorbed from Cdx2 transgenic mouse stomach with intestinal metaplasia. Finally we generated mice whose intestine was extensively excised. Cdx2 transgenic mice with intestinal metaplasia survived even after extensive intestinal excision. We successfully demonstrated that Cdx2 induced not only morphological but also functional absorptive enterocytes in the intestinal metaplastic mucosa in vivo. Our results suggest that Cdx2 is necessary and sufficient by itself to specify the development of intestinal absorptive enterocytes, whereas other factors which are expressed in the small intestine are not always necessary for the differentiation of functional absorptive enterocytes.